Plants having increased tolerance to herbicides

ABSTRACT

The present invention refers to a plant or plant part comprising a polynucleotide encoding a mutated PPO polypeptide, the expression of said polynucleotide confers to the plant or plant part tolerance to herbicides.

This application is a National Stage application of International Application No. PCT/EP2017/083244, filed Dec. 18, 2017, which claims priority to European Patent Application No. 16205383.9, filed on Dec. 20, 2016.

INCORPORATION BY REFERENCE OF MATERIAL SUBMITTED ELECTRONICALLY

The Sequence Listing, which is a part of the present disclosure, is submitted concurrently with the specification as a text file. The name of the text file containing the Sequence Listing is “78939_Seqlisting.txt”, which was created on Mar. 21, 2019 and is 518,123 bytes in size. The subject matter of the Sequence Listing is incorporated herein in its entirety by reference.

FIELD OF THE INVENTION

The present invention relates in general to methods for conferring on plants agricultural level tolerance to a herbicide. Particularly, the invention refers to plants having an increased tolerance to PPO-inhibiting herbicides. More specifically, the present invention relates to methods and plants obtained by mutagenesis and cross-breeding and transformation that have an increased tolerance to PPO-inhibiting herbicides.

BACKGROUND OF THE INVENTION

Herbicides that inhibit protoporphyrinogen oxidase (hereinafter referred to as Protox or PPO; EC:1.3.3.4), a key enzyme in the biosynthesis of protoporphyrin IX, have been used for selective weed control since the 1960s. PPO catalyzes the last common step in chlorophyll and heme biosynthesis which is the oxidation of protoporphyrinogen IX to protoporphyrin IX. (Matringe et al. 1989. Biochem. 1. 260: 231). PPO-inhibiting herbicides include many different structural classes of molecules (Duke et al. 1991. Weed Sci. 39: 465; Nandihalli et al. 1992. Pesticide Biochem. Physiol. 43: 193; Matringe et al. 1989. FEBS Lett. 245: 35; Yanase and Andoh. 1989. Pesticide Biochem. Physiol. 35: 70). These herbicidal compounds include the diphenylethers {e.g. lactofen, (+−)-2-ethoxy-1-methyl-2-oxoethyl 5-{2-chloro-4-(trifluoromethyl)phenoxy}-2-nitrobenzoate; acifluorfen, 5-{2-chloro-4-(trifluoromethyl)phenoxy}-2-nitrobenzoic acid; its methyl ester; or oxyfluorfen, 2-chloro-1-(3-ethoxy-4-nitrophenoxy)-4-(trifluorobenzene)}, oxidiazoles, (e.g. oxidiazon, 3-{2,4-dichloro-5-(1-methylethoxy)phenyl}-5-(1,1-dimethylethyl)-1,3,4-oxadiazol-2-(3H)-one), cyclic imides (e.g. S-23142, N-(4-chloro-2-fluoro-5-propargyloxyphenyl)-3,4,5,6-tetrahydrophthalimide; chlorophthalim, N-(4-chlorophenyl)-3,4,5,6-tetrahydrophthalimide), phenyl pyrazoles (e.g. TNPP-ethyl, ethyl 2-{1-(2,3,4-trichlorophenyl)-4-nitropyrazolyl-5-oxy}propionate; M&B 39279), pyridine derivatives (e.g. LS 82-556), and phenopylate and its O-phenylpyrrolidino- and piperidinocarbamate analogs. Many of these compounds competitively inhibit the normal reaction catalyzed by the enzyme, apparently acting as substrate analogs.

Application of PPO-inhibiting herbicides results in the accumulation of protoporphyrinogen IX in the chloroplast and mitochondria, which is believed to leak into the cytosol where it is oxidized by a peroxidase. When exposed to light, protoporphyrin IX causes formation of singlet oxygen in the cytosol and the formation of other reactive oxygen species, which can cause lipid peroxidation and membrane disruption leading to rapid cell death (Lee et al. 1993. Plant Physiol. 102: 881).

Not all PPO enzymes are sensitive to herbicides which inhibit plant PPO enzymes. Both the Escherichia coli and Bacillus subtilis PPO enzymes (Sasarmen et al. 1993. Can. J. Microbiol. 39: 1155; Dailey et al. 1994. J. Biol. Chem. 269: 813) are resistant to these herbicidal inhibitors. Mutants of the unicellular alga Chlamydomonas reinhardtii resistant to the phenylimide herbicide S-23142 have been reported (Kataoka et al. 1990. J. Pesticide Sci. 15: 449; Shibata et al. 1992. In Research in Photosynthesis, Vol. III, N. Murata, ed. Kluwer:Netherlands. pp. 567-70). At least one of these mutants appears to have an altered PPO activity that is resistant not only to the herbicidal inhibitor on which the mutant was selected, but also to other classes of protox inhibitors (Oshio et al. 1993. Z. Naturforsch. 48c: 339; Sato et al. 1994. In ACS Symposium on Porphyric Pesticides, S. Duke, ed. ACS Press: Washington, D.C.). A mutant tobacco cell line has also been reported that is resistant to the inhibitor S-21432 (Che et al. 1993. Z. Naturforsch. 48c: 350). Auxotrophic E. coli mutants have been used to confirm the herbicide resistance of cloned plant PPO-inhibiting herbicides.

Three main strategies are available for making plants tolerant to herbicides, i.e. (1) detoxifying the herbicide with an enzyme which transforms the herbicide, or its active metabolite, into non-toxic products, such as, for example, the enzymes for tolerance to bromoxynil or to basta (EP242236, EP337899); (2) mutating the target enzyme into a functional enzyme which is less sensitive to the herbicide, or to its active metabolite, such as, for example, the enzymes for tolerance to glyphosate (EP293356, Padgette S. R. et al., J. Biol. Chem., 266, 33, 1991); or (3) overexpressing the sensitive enzyme so as to produce quantities of the target enzyme in the plant which are sufficient in relation to the herbicide, in view of the kinetic constants of this enzyme, so as to have enough of the functional enzyme available despite the presence of its inhibitor. The third strategy was described for successfully obtaining plants which were tolerant to PPO inhibitors (see e.g. U.S. Pat. No. 5,767,373 or U.S. Pat. No. 5,939,602, and patent family members thereof.). In addition, US 2010/0100988 and WO 2007/024739 discloses nucleotide sequences encoding amino acid sequences having enzymatic activity such that the amino acid sequences are resistant to PPO inhibitor herbicidal chemicals, in particular 3-phenyluracil inhibitor specific PPO mutants.

WO 2012/080975 discloses plants the tolerance of which to a PPO-inhibiting herbicide. In particular, WO 2012/080975 discloses that the introduction of nucleic acids which code for a mutated PPO of an Amaranthus type II PPO in which the Arginine at position 128 had been replaced by a leucine, alanine, or valine, and the phenylalanine at position 420 had been replaced by a methionine, cysteine, isoleucine, leucine, or threonine, confers increased tolerance/resistance to a benzoxazinone-derivative herbicide. WO 2013/189984 describes that the introduction of nucleic acids which code for a mutated PPO in which the leucine corresponding to position 397, and the phenylalanine corresponding to position 420 of an Amaranthus type II PPO are replaced confers increased tolerance/resistance to PPO inhibiting herbicides. WO2015/022636 describes novel PPO mutants in which which the arginine corresponding to position 128, and the phenylalanine corresponding to position 420 of an Amaranthus type II PPO are replaced by amino acids which are different from those disclosed by WO 2012/080975. WO2015/022640 discloses novel PPO enzymes and mutants thereof derived from Alopecurus myosuroides. WO2015/092706 describes that the transfer of mutations from Amaranthus (as described above) into crop PPO sequences show an increased tolerance effect in plant. U.S. Pat. No. 7,671,254 describes mutated PPO of an Amaranthus type II PPO in which the glycine at positions 210 and/or 211 are deleted. In contrast to positions corresponding to 128, 397, and 420, said positions 210 and/or 211 are known to lie outside the catalytice site of PPO (“non-active sites”). The inventors or the present invention have now surprisingly found out that a substitution of amino acids at Gly210/211, or at other non-active sites, in particular when combined with a substitution at positions at active sites, i.e. corresponding to positions 128, 397, and/or 420 of the Amaranthus type II PPO increase herbicide tolerance.

SUMMARY OF THE INVENTION

Accordingly, in one aspect, the present invention provides a plant or plant part comprising a polynucleotide encoding a mutated PPO polypeptide, the expression of said polynucleotide confers to the plant or plant part tolerance to herbicides.

In a preferred embodiment, said mutated PPO polypeptide comprises one or more of the following motifs.

-   -   i) Motif 1:         -   GT[C/S]GGDP (SEQ ID NO: 131)         -   Wherein the glycine at position 4, and/or 5 within said             motif of the corresponding wildtype sequence is substituted             by any other amino acid     -   ii) Motif 2:         -   [A/S/C]PS[D/N][X][X]L (SEQ ID NO: 132)         -   Wherein the serine at position 3 within said motif of the             corresponding wildtype sequence is substituted by any other             amino acid     -   iii) Motif 3: (SEQ ID NO: 133)         -   [R/Q][E/D]KQQ[L/Y]P         -   Wherein the glutamine at position 4 within said motif of the             corresponding wildtype sequence is substituted by any other             amino acid     -   iv) Motif 4: (SEQ ID NO: 134)         -   L[I/V]PSKE         -   wherein the serine at position 4 within said motif of the             corresponding wildtype sequence is substituted by any other             amino acid.

Preferably, said mutated PPO polypeptide in addition comprises one or more of the following motifs

-   -   a. Motif 5: SQ[N/K/H]KRYI (SEQ ID NO: 135), wherein the Arg at         position 5 within said motif is substituted by any other amino         acid;     -   b. Motif 6: TLGTLFSS (SEQ ID NO: 136), wherein the Leu at         position 2 within said motif is substituted by by any other         amino acid;     -   c. Motif 7: [F/Y]TTF[V/I]GG (SEQ ID NO: 137), wherein the Phe at         position 4 within said motif is substituted by by any other         amino acid.

In some aspects, the present invention provides a seed capable of germination into a plant comprising in at least some of its cells a polynucleotide operably linked to a promoter operable in plant cells, the promoter capable of expressing a mutated PPO polypeptide encoded by the polynucleotide of the present invention, the expression of the mutated PPO polypeptide conferring to the plant tolerance to herbicides.

In one aspect, the present invention provides a plant cell capable of regenerating a plant comprising in at least some of its cells a polynucleotide operably linked to a promoter operable in plant cells, the promoter capable of expressing a mutated PPO polypeptide encoded by the polynucleotide of the present invention, the expression of the mutated PPO polypeptide conferring to the plant tolerance to herbicides, wherein the plant cell comprises the polynucleotide operably linked to a promoter.

In another aspect, the present invention provides a plant cell comprising a polynucleotide operably linked to a promoter operable in a cell, the promoter capable of expressing a mutated PPO polypeptide encoded by the polynucleotide of the present invention, the expression of the mutated PPO polypeptide conferring to the plant tolerance to herbicides.

In other aspects, the present invention provides a plant product prepared from a plant or plant part comprising in at least some of its cells a polynucleotide operably linked to a promoter operable in plant cells, the promoter capable of expressing a mutated PPO polypeptide encoded by the polynucleotide of the present invention, the expression of the mutated PPO polypeptide conferring to the plant tolerance to herbicides.

In some aspects, the present invention provides a progeny or descendant plant derived from a plant comprising in at least some of its cells a polynucleotide operably linked to a promoter operable in plant cells, the promoter capable of expressing a mutated PPO polypeptide encoded by the polynucleotide of the present invention, wherein the progeny or descendant plant comprises in at least some of its cells the recombinant polynucleotide operably linked to the promoter, the expression of the mutated PPO polypeptide conferring to the progeny or descendant plant tolerance to the herbicides.

In other aspects, the present invention provides a method for controlling weeds at a locus for growth of a plant, the method comprising: (a) applying an herbicide composition comprising herbicides to the locus; and (b) planting a seed at the locus, wherein the seed is capable of producing a plant that comprises in at least some of its cells a polynucleotide operably linked to a promoter operable in plant cells, the promoter capable of expressing a mutated PPO polypeptide encoded by the polynucleotide of the present invention, the expression of the mutated PPO polypeptide conferring to the plant tolerance to herbicides.

In some aspects, the present invention provides a method for controlling weeds at a locus for growth of a plant, the method comprising: applying an herbicidal composition comprising herbicides to the locus; wherein said locus is: (a) a locus that contains: a plant or a seed capable of producing said plant; or (b) a locus that is to be after said applying is made to contain the plant or the seed; wherein the plant or the seed comprises in at least some of its cells a polynucleotide operably linked to a promoter operable in plant cells, the promoter capable of expressing a mutated PPO polypeptide encoded by the polynucleotide of the present invention, the expression of the mutated PPO polypeptide conferring to the plant tolerance to herbicides.

In one aspect, step (a) occurs before, after, or concurrently with step (b).

In other aspects, the present invention provides a method of producing a plant having tolerance to herbicides, the method comprising regenerating a plant from a plant cell transformed with a polynucleotide operably linked to a promoter operable in plant cells, the promoter capable of expressing a mutated PPO polypeptide encoded by the polynucleotide of the present invention, the expression of the mutated PPO polypeptide conferring to the plant tolerance to herbicides.

In one aspect, the present invention provides a method of producing a progeny plant having tolerance to herbicides, the method comprising: crossing a first herbicide-tolerant plant with a second plant to produce a herbicide-tolerant progeny plant, wherein the first plant and the progeny plant comprise in at least some of their cells a polynucleotide operably linked to a promoter operable in plant cells, the promoter capable of expressing a mutated PPO polypeptide encoded by the polynucleotide of the present invention, the expression of the mutated PPO polypeptide conferring to the plant tolerance to herbicides.

In addition, the present invention refers to a method for identifying a herbicide by using a mutated PPO of the present invention.

Said method comprises the steps of:

-   a) generating a transgenic cell or plant comprising a nucleic acid     encoding a mutated PPO of the present invention, wherein the mutated     PPO of the present invention is expressed; -   b) applying a herbicide to the transgenic cell or plant of a) and to     a control cell or plant of the same variety; -   c) determining the growth or the viability of the transgenic cell or     plant and the control cell or plant after application of said test     compound, and -   d) selecting test compounds which confer reduced growth to the     control cell or plant as compared to the growth of the transgenic     cell or plant.

Another object refers to a method of identifying a nucleotide sequence encoding a mutated PPO of the present invention which is resistant or tolerant to a herbicide, the method comprising:

-   a) generating a library of mutated PPO-encoding nucleic acids, -   b) screening a population of the resulting mutated PPO-encoding     nucleic acids by expressing each of said nucleic acids in a cell or     plant and treating said cell or plant with a herbicide, -   c) comparing the herbicide-tolerance levels provided by said     population of mutated PPO encoding nucleic acids with the     herbicide-tolerance level provided by a control PPO-encoding nucleic     acid, -   d) selecting at least one mutated PPO-encoding nucleic acid that     provides a significantly increased level of tolerance to a herbicide     as compared to that provided by the control PPO-encoding nucleic     acid.

In a preferred embodiment, the mutated PPO-encoding nucleic acid selected in step d) provides at least 2-fold as much tolerance to a herbicide as compared to that provided by the control PPO-encoding nucleic acid.

The resistance or tolerance can be determined by generating a transgenic plant comprising a nucleic acid sequence of the library of step a) and comparing said transgenic plant with a control plant.

Another embodiment refers to an isolated and/or recombinantly produced and/or synthetic nucleic acid molecule comprising a nucleic acid molecule encoding a mutated PPO polypeptide selected from the group consisting of:

-   -   (a) a nucleic acid molecule encoding a mutated PPO polypeptide         comprising the sequence of-SEQ ID NO: 1, 2, 3, 4, 5, 6, 7, 8, 9,         10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25,         26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41,         42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57,         58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73,         74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89,         90, 91, 92, 93, 94, 95, 96, 97, 98, 99, 100, 101, 102, 103, 104,         105, 106, 107, 108, 109, 110, 111, 112, 113, 114, 115, 116, 117,         or 129, or a variant, paralogue, orthologue or homolog thereof;     -   (b) a nucleic acid molecule, which, as a result of the         degeneracy of the genetic code, can be derived from a nucleic         acid molecule encoding a mutated PPO polypeptide comprising the         sequence of-SEQ ID NO: 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12,         13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28,         29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44,         45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60,         61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76,         77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92,         93, 94, 95, 96, 97, 98, 99, 100, 101, 102, 103, 104, 105, 106,         107, 108, 109, 110, 111, 112, 113, 114, 115, 116, 117, or 129,         or a variant, paralogue, orthologue or homolog thereof, and         confers increased herbicide tolerance or resistance, as compared         to a corresponding, e.g. non-transformed, wild type plant cell,         a plant or a part thereof;     -   (c) a nucleic acid molecule encoding a mutated PPO polypeptide         having 30% or more identity, preferably at least 40%, 50%, 60%,         70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, 99,5% or more,         with the amino acid sequence of the PPO polypeptide sequence of         SEQ ID NO: 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15,         16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31,         32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47,         48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63,         64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79,         80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95,         96, 97, 98, 99, 100, 101, 102, 103, 104, 105, 106, 107, 108,         109, 110, 111, 112, 113, 114, 115, 116, 117, or 129, and confers         increased herbicide tolerance or resistance, as compared to a         corresponding, e.g. non-transformed, wild type plant cell, a         plant or a part thereof;     -   (d) nucleic acid molecule which hybridizes with a nucleic acid         molecule of (a), (b), or (c), under stringent hybridization         conditions and confers increased herbicide tolerance or         resistance, as compared to a corresponding, e.g.         non-transformed, wild type plant cell, a plant or a part         thereof;     -    wherein the amino acid sequence of the encoded mutated PPO         polypeptide differs from the wildtype amino acid sequence of a         PPO polypeptide at one or more positions corresponding to         positions 32, 53, 57, 61, 63, 64, 65, 67, 71, 76, 82, 83, 85,         86, 87, 88, 91, 103, 104, 106, 108, 116, 119, 126, 127, 129,         139, 159, 210, 211, 224, 245, 246, 248, 249, 252, 253, 254, 255,         257, 259, 260, 262, 264, 286, 291, 305, 308, 309, 323, 335, 343,         345, 358, 372, 373, 387, 391, 392, 400, 412, 414, 415, 425, 428,         431, 433, 434, 435, 436, 447, 451, 453, 464, 466, 482 of SEQ ID         NO: 1 or 2, wherein said difference refers to a substitution of         the amino acid at that positions by any other amino acid.

Another embodiment refers to an isolated and/or recombinantly produced and/or synthetic nucleic acid molecule comprising a nucleic acid molecule encoding a mutated PPO polypeptide selected from the group consisting of:

-   -   (a) a nucleic acid molecule encoding a mutated PPO polypeptide         comprising the sequence of-SEQ ID NO: 1, 2, 3, 4, 5, 6, 7, 8, 9,         10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25,         26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41,         42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57,         58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73,         74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89,         90, 91, 92, 93, 94, 95, 96, 97, 98, 99, 100, 101, 102, 103, 104,         105, 106, 107, 108, 109, 110, 111, 112, 113, 114, 115, 116, 117,         or 129, or a variant, paralogue, orthologue or homolog thereof;     -   (b) a nucleic acid molecule, which, as a result of the         degeneracy of the genetic code, can be derived from a nucleic         acid molecule encoding a mutated PPO polypeptide comprising the         sequence of-SEQ ID NO: 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12,         13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28,         29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44,         45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60,         61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76,         77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92,         93, 94, 95, 96, 97, 98, 99, 100, 101, 102, 103, 104, 105, 106,         107, 108, 109, 110, 111, 112, 113, 114, 115, 116, 117, or 129,         or a variant, paralogue, orthologue or homolog thereof, and         confers increased herbicide tolerance or resistance, as compared         to a corresponding, e.g. non-transformed, wild type plant cell,         a plant or a part thereof;     -   (c) a nucleic acid molecule encoding a mutated PPO polypeptide         having 30% or more identity, preferably at least 40%, 50%, 60%,         70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, 99,5% or more,         with the amino acid sequence of the PPO polypeptide sequence of         SEQ ID NO: 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15,         16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31,         32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47,         48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63,         64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79,         80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95,         96, 97, 98, 99, 100, 101, 102, 103, 104, 105, 106, 107, 108,         109, 110, 111, 112, 113, 114, 115, 116, 117, or 129, and confers         increased herbicide tolerance or resistance, as compared to a         corresponding, e.g. non-transformed, wild type plant cell, a         plant or a part thereof;     -   (d) nucleic acid molecule which hybridizes with a nucleic acid         molecule of (a), (b), or (c), under stringent hybridization         conditions and confers increased herbicide tolerance or         resistance, as compared to a corresponding, e.g.         non-transformed, wild type plant cell, a plant or a part         thereof;     -    wherein the amino acid sequence of the encoded mutated PPO         polypeptide differs from the wildtype amino acid sequence of a         PPO polypeptide at one or more positions corresponding to         positions 40, 250, 391, 520, 521, 522, 523, 524, 525, 526, 527,         528, 529, 530, 531, 532, 533, 534 of SEQ ID NO: 1 or 2, wherein         said difference refers to a deletion or an insertion of the         amino acid at that positions.

Preferably, the difference corresponding to position 391 of SEQ ID NO:1 refers to an insertion, and the difference corresponding to positions 40, 250, 520, 521, 522, 523, 524, 525, 526, 527, 528, 529, 530, 531, 532, 533, 534 of SEQ ID NO: 1 or 2 refers to a deletion.

Another object refers to an expression cassette comprising the nucleic acid molecule of the present invention and a promoter operable in plant cells.

Another object refers to an isolated, recombinant and/or chemically synthesized mutated PPO polypeptide encoded by the nucleic acid molecule as claimed in any of claims 17 to 25 or a polypeptide having at least 80%, 90%, 95%, 98%, 99% identity to the sequence of SEQ ID NO: 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, 100, 101, 102, 103, 104, 105, 106, 107, 108, 109, 110, 111, 112, 113, 114, 115, 116, 117, or 129, or a variant, paralogue, orthologue or homolog thereof, wherein the amino acid sequence of the mutated PPO polypeptide differs from the wildtype amino acid sequence of a PPO polypeptide at one or more positions corresponding to positions 32, 53, 57, 61, 63, 64, 65, 67, 71, 76, 82, 83, 85, 86, 87, 88, 91, 103, 104, 106, 108, 116, 119, 126, 127, 129, 139, 159, 210, 211, 224, 245, 246, 248, 249, 252, 253, 254, 255, 257, 259, 260, 262, 264, 286, 291, 305, 308, 309, 323, 335, 343, 345, 358, 372, 373, 387, 391, 392, 400, 412, 414, 415, 425, 428, 431, 433, 434, 435, 436, 447, 451, 453, 464, 466, 482 of SEQ ID NO: 1 or 2, wherein said difference refers to a substitution of the amino acid at that positions by any other amino acid.

Another object refers to an isolated, recombinant and/or chemically synthesized mutated PPO polypeptide encoded by the nucleic acid molecule as claimed in any of claims 17 to 25 or a polypeptide having at least 80%, 90%, 95%, 98%, 99% identity to the sequence of SEQ ID NO: 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, 100, 101, 102, 103, 104, 105, 106, 107, 108, 109, 110, 111, 112, 113, 114, 115, 116, 117, or 129, or a variant, paralogue, orthologue or homolog thereof, wherein the amino acid sequence of the mutated PPO polypeptide differs from the wildtype amino acid sequence of a PPO polypeptide at one or more positions corresponding to positions 40, 250, 391, 520, 521, 522, 523, 524, 525, 526, 527, 528, 529, 530, 531, 532, 533, 534 of SEQ ID NO: 1 or 2, wherein said difference refers to a deletion or an insertion of the amino acid at that positions.

In still further aspects, the present invention provides a plant or plant part comprising in at least some of its cells a polynucleotide operably linked to a promoter operable in plant cells, the promoter capable of expressing a mutated PPO polypeptide encoded by the polynucleotide, the expression of the mutated PPO polypeptide conferring to the plant tolerance to herbicides, wherein the plant or plant part further exhibits a second or third herbicide-tolerant trait.

In another embodiment, the invention refers to a plant cell transformed by and expressing a mutated PPO nucleic acid according to the present invention or a plant which has been mutated to obtain a plant expressing, preferably over-expressing a mutated PPO nucleic acid according to the present invention, wherein expression of said nucleic acid in the plant cell results in increased resistance or tolerance to a herbicide as compared to a wild type variety of the plant cell

In another embodiment, the invention refers to a plant comprising a plant cell according to the present invention, wherein expression of the nucleic acid in the plant results in the plant's increased resistance to herbicide as compared to a wild type variety of the plant.

The plants of the present invention can be transgenic or non-transgenic.

Preferably, the expression of the nucleic acid of the invention in the plant results in the plant's increased resistance to herbicides as compared to a wild type variety of the plant.

In another embodiment, the invention refers to a seed produced by a transgenic plant comprising a plant cell of the present invention, wherein the seed is true breeding for an increased resistance to a herbicide as compared to a wild type variety of the seed.

In another embodiment, the invention refers to a method of producing a transgenic plant cell with an increased resistance to a herbicide as compared to a wild type variety of the plant cell comprising, transforming the plant cell with an expression cassette comprising a polynucleotide operably linked to a promoter operable in plant cells, the promoter capable of expressing a mutated PPO polypeptide encoded by the polynucleotide. In another embodiment, the invention refers to a method of producing a transgenic plant comprising, (a) transforming a plant cell with an expression cassette comprising a polynucleotide operably linked to a promoter operable in plant cells, the promoter capable of expressing a mutated PPO polypeptide encoded by the polynucleotide, and (b) generating a plant with an increased resistance to herbicide from the plant cell.

Preferably, the expression cassette further comprises a transcription initiation regulatory region and a translation initiation regulatory region that are functional in the plant.

LEGEND TO FIGURES

FIG. 1 shows a germination assay using transgenic Arabidopsis plants (SEQ ID NO:1 mutated AMATU_PPO2_G211A_F420M). Plants were treated with the indicated concentrations of Saflufenacil. Pictures were taken 14 days after treatment.

FIG. 2 shows T1 Tolerance Spray using transgenic Arabidopsis plants (SEQ ID NO:1 mutated AMATU_PPO2_G211A_F420M). Pictures were taken 8 days after treatment (days after spraying). Top at bottom contain 2 wild type plants, upper 5 pots contain independent transgenic events (T1 plants, selected by confirming presence of resistance gene AHAS).

FIG. 3 shows Transgenic Arabidopsis plants sprayed post in the greenhouse with the indicated amount of Saflufenacil+1% (v/v) MSO. Plants harbor the SEQ ID NO:1 mutated traits: A) AMATU_PPO2_R128A_G210S_F420M, B) AMATU_PPO2_R128A_S387L_F420M, C) AMATU_PPO2_G211A_L397QF420M, D) AMATU_PPO2_R128A_F420M E) AMATU_PPO2_F420V F) AMATU_PPO2_L397Q_F420M G) Wild-type (non-transgenic). Evaluation performed 6 Days After Treatment (DAT).

FIG. 4 shows transgenic Arabidopsis plants sprayed post in the greenhouse with the indicated amount of Trifludimoxazine+1% (v/v) MSO. Plants harbor the SEQ ID NO:1 mutated traits: A) AMATU_PPO2_R128A_G210S_F420M, B) AMATU_PPO2_R128A_S387L_F420M, C) AMATU_PPO2_G211A_F420V, D) AMATU_PPO2_G211A_L397Q_F420M, E) AMATU_PPO2_R128A_F420M F) AMATU_PPO2_F420V G) AMATU_PPO2_L397Q_F420M H) Wild-type (non-transgenic). Evaluation performed 6 Days After Treatment (DAT).

FIG. 5 shows transgenic Arabidopsis plants sprayed post in the greenhouse with the indicated amount of Saflufenacil+Trifludimoxazine+1% (v/v) MSO. Plants harbor the SEQ ID NO:1 mutated traits: A) AMATU_PPO2_R128A_G210S_F420M, B) AMATU_PPO2_R128A_S387L_F420M, C) AMATU_PPO2_G211A_F420V, D) AMATU_PPO2_G211A_L397Q_F420M , E) AMATU_PPO2_R128A_F420M F) AMATU_PPO2_F420V G) AMATU_PPO2_L397Q_F420M H) Wild-type (non-transgenic). Evaluation performed 6 Days After Treatment (DAT).

FIG. 6 shows transgenic Arabidopsis plants sprayed post in the greenhouse with the indicated amount of Uracilpyridine 2+1% (v/v) MSO. Plants harbor the SEQ ID NO:1 mutated traits: A) AMATU_PPO2_R128A_G210S_F420M, B) AMATU_PPO2_R128A_S387L_F420M, C) AMATU_PPO2_G211A_F420V, D) AMATU_PPO2_G211A_L397Q_F420M , E) AMATU_PPO2_R128A_F420M F) AMATU_PPO2_F420V G) AMATU_PPO2_L397Q_F420M H) Wild-type (non-transgenic). Evaluation performed 6 Days After Treatment (DAT).

FIG. 7 shows transgenic Arabidopsis plants sprayed post in the greenhouse with the indicated amount of Uracilpyridine 4+1% (v/v) MSO. Plants harbor the SEQ ID NO:1 mutated traits: A) AMATU_PPO2_R128A_G210S_F420M, B) AMATU_PPO2_R128A_S387L_F420M, C) AMATU_PPO2_G211A_F420V, D) AMATU_PPO2_G211A_L397Q_F420M , E) AMATU_PPO2_R128A_F420M F) AMATU_PPO2_F420V G) AMATU_PPO2_L397Q_F420M H) Wild-type (non-transgenic). Evaluation performed 6 Days After Treatment (DAT).

FIG. 8 shows transgenic Arabidopsis plants sprayed post in the greenhouse with the indicated amount of Uracilpyridine 1+1% (v/v) MSO. Plants harbor the SEQ ID NO:1 mutated traits: A) AMATU_PPO2_R128A_G210S_F420M, B) AMATU_PPO2_R128A_S387L_F420M, C) AMATU_PPO2_G211A_L397Q_F420M , D) AMATU_PPO2_R128A_F420M E) AMATU_PPO2_F420V F) AMATU_PPO2_L397Q_F420M G) Wild-type (non-transgenic). Evaluation performed 6 Days After Treatment (DAT).

FIG. 9 shows transgenic Arabidopsis plants sprayed post in the greenhouse with the indicated amount of Sulfentrazone+1% (v/v) MSO. Plants harbor the SEQ ID NO:1 mutated traits: A) AMATU_PPO2_R128A_G210S_F420M, B) AMATU_PPO2_R128A_S387L_F420M, C) AMATU_PPO2_G211A_L397Q_F420M , D) AMATU_PPO2_R128A_F420M E) AMATU_PPO2_F420V F) AMATU_PPO2_L397Q_F420M G) Wild-type (non-transgenic). Evaluation performed 6 Days After Treatment (DAT).

FIG. 10 shows transgenic Arabidopsis plants sprayed post in the greenhouse with the indicated amount of Flumioxazin+1% (v/v) MSO. Plants harbor the SEQ ID NO:1 mutated traits: A) AMATU_PPO2_R128A_G210S_F420M, B) AMATU_PPO2_R128A_S387L_F420M, C) AMATU_PPO2_R128A_F420M D) AMATU_PPO2_F420V E) AMATU_PPO2_L397Q_F420M F) Wild-type (non-transgenic). Evaluation performed 6 Days After Treatment (DAT).

FIG. 11 shows transgenic Arabidopsis plants harboring SEQ ID NO:1 mutated AMATU_PPO2_G211A_F420M construct, sprayed post in the greenhouse with the indicated amount of A) Saflufenacil+Trifludimoxazine, B) Tiafenacil, C) Uracilpyridine 6, D) Pyraflufen-ethyl, E) Uracilpyridine 7, with +1% (v/v) MSO. Evaluation performed 14 Days After Treatment (DAT) and is shown as injury (%) relative to non-transgenic treated plants.

FIG. 12 shows examples of transgenic Zea mays harboring AmtuPPX2L variants after pre-emergent treatment with Saflufenacil.

FIG. 13 shows tolerance of transgenic Glycine max harboring AmtuPPX2L variants after treatment with a mixture of Saflufenacil and Trifludimoxazine. 0/0 denotes no active ingredient, 50/25 denotes 50 grams of active ingredient per hectare (Al/Ha) of Saflufenacil and 25 grams Al/Ha of Trifludimoxazine, 100/50 denotes 100 g Al/Ha Saflufenacil and 50 g Al/Ha Trifludimoxazine and 200/100 denotes 200 g Al/Ha Saflufenacil and 100 g Al/Ha Trifludimoxazine. Wild type untransformed is represented by the soy (Glycine ma4 cultivar Jake (cv. Jake).

FIG. 14 shows examples of herbicide tolerance of transgenic Glycine max harboring AmtuPPX2L variants after treatment with a mixture of Saflufenacil and Trifludimoxazine. 0/0 denotes no active ingredient, 25/50 denotes 50 grams of active ingredient per hectare (Al/Ha) of Saflufenacil and 25 grams Al/Ha of Trifludimoxazine, 50/100 denotes 100 g Al/Ha Saflufenacil and 50 g Al/Ha Trifludimoxazine and 100/200 denotes 200 g Al/Ha Saflufenacil and 100 g Al/Ha Trifludimoxazine. Wild type untransformed is represented by the soy (Glycine ma4 cultivar Jake (cv. Jake).

LEGEND TO SEQUENCE LISTING SEQ ID Organism 1 Amaranthus tuberculatus 2 Amaranthus tuberculatus 3 Amaranthus tuberculatus 4 Amaranthus tuberculatus 5 Amaranthus hypochondriacus 6 Amaranthus tuberculatus 7 Spinacia oleracea 8 Vitis vinifera 9 Ricinus communis 10 Theobroma cacao 11 Glycine max 12 Prunus persica 13 Medicago truncatula 14 Fragaria vesca subsp. vesca 15 Citrus clementina 16 Citrus clementina 17 Cicer arietinum 18 Cucumis sativus 19 Cucumis sativus 20 Nicotiana tabacum 21 Solanum lycopersicum 22 Arabidopsis thaliana 23 Arabidopsis lyrata subsp. lyrata 24 Arabidopsis thaliana 25 Arabidopsis thaliana 26 Ambrosia artemisiifolia 27 Setaria italica 28 Sorghum bicolor 29 Arabidopsis thaliana 30 Zea mays 31 Zea mays 32 LEMPA 33 LEMPA 34 Populus trichocarpa 35 Capsella rubella 36 Brachypodium distachyon 37 Oryza sativa Japonica Group 38 Picea sitchensis 39 Solarium tuberosum 40 Oryza sativa Indica Group 41 Oryza sativa Japonica Group 42 Eutrema salsugineum 43 Selaginella moellendorffii 44 Selaginella moellendorffii 45 Amaranthus tuberculatus 46 Amaranthus tuberculatus 47 Zea mays 48 Aegilops tauschii 49 Genlisea aurea 50 Amborella trichopoda 51 Rhodothermus marinus 52 Salinibacter ruber 53 Salinibacter ruber M8 54 Zea mays 55 Rhodothermus marinus 56 Caldithrix abyssi 57 Opitutus terrae PB90-1 58 Verrucomicrobia bacterium 59 Ignavibacterium album 60 Coraliomargarita sp. CAG:312 61 Salisaeta longa 62 Ambrosia artemisiifolia 63 Melioribacter roseus P3M-2 64 Halothiobacillus neapolitanus c2 65 Chondrus crispus 66 Rubritalea marina 67 Acidobacteria bacterium 68 Coraliomargarita akajimensis DSM 45221 69 Oscillochloris trichoides DG6 70 Opitutaceae bacterium TAV1 71 Amborella trichopoda 72 Opitutaceae bacterium TAV5 73 Chloroflexus sp. Y-400-fl 74 Leptospirillum sp. Group II ‘5-way CG’ 75 Leptospirillum ferriphilum ML-04 76 Verrucomicrobia bacterium SCGC AAA300-017 77 Chloroflexus aggregans DSM 9485 78 Desulfurobacterium thermolithotrophum 79 Desulfurobacterium sp. TC5-1 80 Arthrospira platensis C1 81 Leptospirillum sp. Group II ‘C75’ 82 Verrucomicrobiae bacterium DG1235 83 Verrucomicrobia bacterium SCGC AAA300-K03 84 Synechococcus sp. JA-3-3Ab 85 Hymenobacter norwichensis 86 Pontibacter sp. BAB1700 87 Leptospirillum ferrodiazotrophum 88 Prevotella histicola F0411 89 Flexithrix dorotheae 90 Geobacter metallireducens GS-15 91 Synechococcus sp. JA-2-3B′a(2-13) 92 Crinalium epipsammum PCC 9333 93 Planctomyces maris 94 Geobacter uraniireducens Rf4 95 Acidithiobacillus ferrivorans 96 Prevotella melaninogenica 97 Thermovibrio ammonificans 98 Brassica_rapa 99 Brassica_rapa 100 Gossypium 101 Gossypium 102 Conyza_canadensis 103 Conyza_canadensis 104 Kochia_scobaria 105 Lolium_rigidum 106 Lolium_rigidum 107 Gossypium hirsutum PPO1 108 Beta vulgaris PPO1 109 Hordeum vulgare PPO1 110 Hordeum vulgare PPO2 111 Triticum aestivum PPO1 112 Solarium lycopersicum PPO2 113 Triticum aestivum PPO1_v2 114 Gossypium hirsutum PPO1_v2 115 Gossypium hirsutum PPO2 116 Beta vulgaris PPO1_v2 117 Brassica napus_PPO2 129 Alopecurus myosuroides

DETAILED DESCRIPTION

The articles “a” and “an” are used herein to refer to one or more than one (i.e., to at least one) of the grammatical object of the article. By way of example, “an element” means one or more elements.

As used herein, the word “comprising,” or variations such as “comprises” or “comprising,” will be understood to imply the inclusion of a stated element, integer or step, or group of elements, integers or steps, but not the exclusion of any other element, integer or step, or group of elements, integers or steps.

The term “control of undesired vegetation or weeds” is to be understood as meaning the killing of weeds and/or otherwise retarding or inhibiting the normal growth of the weeds. Weeds, in the broadest sense, are understood as meaning all those plants which grow in locations where they are undesired. The weeds of the present invention include, for example, dicotyledonous and monocotyledonous weeds. Dicotyledonous weeds include, but are not limited to, weeds of the genera: Sinapis, Lepidium, Galium, Stellaria, Matricaria, Anthemis, Galinsoga, Chenopodium, Urtica, Senecio, Amaranthus, Portulaca, Xanthium, Convolvulus, Ipomoea, Polygonum, Sesbania, Ambrosia, Cirsium, Carduus, Sonchus, Solanum, Rorippa, Rotala, Lindernia, Lamium, Veronica, Abutilon, Emex, Datura, Viola, Galeopsis, Papaver, Centaurea, Trifolium, Ranunculus, and Taraxacum. Monocotyledonous weeds include, but are not limited to, weeds of of the genera: Echinochloa, Setaria, Panicum, Digitaria, Phleum, Poa, Festuca, Eleusine, Brachiaria, Lolium, Bromus, Avena, Cyperus, Sorghum, Agropyron, Cynodon, Monochoria, Fimbristyslis, Sagittaria, Eleocharis, Scirpus, Paspalum, Ischaemum, Sphenoclea, Dactyloctenium, Agrostis, Alopecurus, and Apera. In addition, the weeds of the present invention can include, for example, crop plants that are growing in an undesired location. For example, a volunteer maize plant that is in a field that predominantly comprises soybean plants can be considered a weed, if the maize plant is undesired in the field of soybean plants.

The term “plant” is used in its broadest sense as it pertains to organic material and is intended to encompass eukaryotic organisms that are members of the Kingdom Plantae, examples of which include but are not limited to vascular plants, vegetables, grains, flowers, trees, herbs, bushes, grasses, vines, ferns, mosses, fungi and algae, etc, as well as clones, offsets, and parts of plants used for asexual propagation (e.g. cuttings, pipings, shoots, rhizomes, underground stems, clumps, crowns, bulbs, corms, tubers, rhizomes, plants/tissues produced in tissue culture, etc.). The term “plant” further encompasses whole plants, ancestors and progeny of the plants and plant parts, including seeds, shoots, stems, leaves, roots (including tubers), flowers, florets, fruits, pedicles, peduncles, stamen, anther, stigma, style, ovary, petal, sepal, carpel, root tip, root cap, root hair, leaf hair, seed hair, pollen grain, microspore, cotyledon, hypocotyl, epicotyl, xylem, phloem, parenchyma, endosperm, a companion cell, a guard cell, and any other known organs, tissues, and cells of a plant, and tissues and organs, wherein each of the aforementioned comprise the gene/nucleic acid of interest. The term “plant” also encompasses plant cells, suspension cultures, callus tissue, embryos, meristematic regions, gametophytes, sporophytes, pollen and microspores, again wherein each of the aforementioned comprises the gene/nucleic acid of interest.

Plants that are particularly useful in the methods of the invention include all plants which belong to the superfamily Viridiplantae, in particular monocotyledonous and dicotyledonous plants including fodder or forage legumes, ornamental plants, food crops, trees or shrubs selected from the list comprising Acer spp., Actinidia spp., Abelmoschus spp., Agave sisalana, Agropyron spp., Agrostis stolonifera, Allium spp., Amaranthus spp., Ammophila arenaria, Ananas comosus, Annona spp., Apium graveolens, Arachis spp, Artocarpus spp., Asparagus officinalis, Avena spp. (e.g. Avena sativa, Avena fatua, Avena byzantina, Avena fatua var. sativa, Avena hybrida), Averrhoa carambola, Bambusa sp., Benincasa hispida, Bertholletia excelsea, Beta vulgaris, Brassica spp. (e.g. Brassica napus, Brassica rapa ssp. [canola, oilseed rape, turnip rape]), Cadaba farinosa, Camellia sinensis, Canna indica, Cannabis sativa, Capsicum spp., Carex elata, Carica papaya, Carissa macrocarpa, Carya spp., Carthamus tinctorius, Castanea spp., Ceiba pentandra, Cichorium endivia, Cinnamomum spp., Citrullus lanatus, Citrus spp., Cocos spp., Coffea spp., Colocasia esculenta, Cola spp., Corchorus sp., Coriandrum sativum, Corylus spp., Crataegus spp., Crocus sativus, Cucurbita spp., Cucumis spp., Cynara spp., Daucus carota, Desmodium spp., Dimocarpus longan, Dioscorea spp., Diospyros spp., Echinochloa spp., Elaeis (e.g. Elaeis guineensis, Elaeis oleifera), Eleusine coracana, Eragrostis tef, Erianthus sp., Eriobotrya japonica, Eucalyptus sp., Eugenia uniflora, Fagopyrum spp., Fagus spp., Festuca arundinacea, Ficus carica, Fortunella spp., Fragaria spp., Ginkgo biloba, Glycine spp. (e.g. Glycine max, Soja hispida or Soja max), Gossypium hirsutum, Helianthus spp. (e.g. Helianthus annuus), Hemerocallis fulva, Hibiscus spp., Hordeum spp. (e.g. Hordeum vulgare), Ipomoea batatas, Juglans spp., Lactuca sativa, Lathyrus spp., Lens culinaris, Linum usitatissimum, Litchi chinensis, Lotus spp., Luffa acutangula, Lupinus spp., Luzula sylvatica, Lycopersicon spp. (e.g. Lycopersicon esculentum, Lycopersicon lycopersicum, Lycopersicon pyriforme), Macrotyloma spp., Malus spp., Malpighia emarginata, Mammea americana, Mangifera indica, Manihot spp., Manilkara zapota, Medicago sativa, Melilotus spp., Mentha spp., Miscanthus sinensis, Momordica spp., Morus nigra, Musa spp., Nicotiana spp., Olea spp., Opuntia spp., Ornithopus spp., Oryza spp. (e.g. Oryza sativa, Oryza latifolia), Panicum miliaceum, Panicum virgatum, Passiflora edulis, Pastinaca sativa, Pennisetum sp., Persea spp., Petroselinum crispum, Phalaris arundinacea, Phaseolus spp., Phleum pratense, Phoenix spp., Phragmites australis, Physalis spp., Pinus spp., Pistacia vera, Pisum spp., Poa spp., Populus spp., Prosopis spp., Prunus spp., Psidium spp., Punica granatum, Pyrus communis, Quercus spp., Raphanus sativus, Rheum rhabarbarum, Ribes spp., Ricinus communis, Rubus spp., Saccharum spp., Salix sp., Sambucus spp., Secale cereale, Sesamum spp., Sinapis sp., Solanum spp. (e.g. Solanum tuberosum, Solanum integrifolium or Solanum lycopersicum), Sorghum bicolor, Spinacia spp., Syzygium spp., Tagetes spp., Tamarindus indica, Theobroma cacao, Trifolium spp., Tripsacum dactyloides, Triticosecale rimpaui, Triticum spp. (e.g. Triticum aestivum, Triticum durum, Triticum turgidum, Triticum hybernum, Triticum macha, Triticum sativum, Triticum monococcum or Triticum vulgare), Tropaeolum minus, Tropaeolum majus, Vaccinium spp., Vicia spp., Vigna spp., Viola odorata, Vitis spp., Zea mays, Zizania palustris, Ziziphus spp., amaranth, artichoke, asparagus, broccoli, Brussels sprouts, cabbage, canola, carrot, cauliflower, celery, collard greens, flax, kale, lentil, oilseed rape, okra, onion, potato, rice, soybean, strawberry, sugar beet, sugar cane, sunflower, tomato, squash, tea and algae, amongst others. According to a preferred embodiment of the present invention, the plant is a crop plant. Examples of crop plants include inter alia soybean, sunflower, canola, alfalfa, rapeseed, cotton, tomato, potato or tobacco. Further preferebly, the plant is a monocotyledonous plant, such as sugarcane. Further preferably, the plant is a cereal, such as rice, maize, wheat, barley, millet, rye, sorghum or oats.

Generally, the term “herbicide” is used herein to mean an active ingredient that kills, controls or otherwise adversely modifies the growth of plants. The preferred amount or concentration of the herbicide is an “effective amount” or “effective concentration.” By “effective amount” and “effective concentration” is intended an amount and concentration, respectively, that is sufficient to kill or inhibit the growth of a similar, wild-type, plant, plant tissue, plant cell, or host cell, but that said amount does not kill or inhibit as severely the growth of the herbicide-resistant plants, plant tissues, plant cells, and host cells of the present invention. Typically, the effective amount of a herbicide is an amount that is routinely used in agricultural production systems to kill weeds of interest. Such an amount is known to those of ordinary skill in the art. Herbicidal activity is exhibited by herbicides useful for the the present invention when they are applied directly to the plant or to the locus of the plant at any stage of growth or before planting or emergence. The effect observed depends upon the plant species to be controlled, the stage of growth of the plant, the application parameters of dilution and spray drop size, the particle size of solid components, the environmental conditions at the time of use, the specific compound employed, the specific adjuvants and carriers employed, the soil type, and the like, as well as the amount of chemical applied. These and other factors can be adjusted as is known in the art to promote non-selective or selective herbicidal action. Generally, the herbicide treatments can be applied PPI (Pre Plant Incorporated), PPSA (Post plant surface applied), PRE- or POST-emergent. Postemergent treatment typically occurs to relatively immature undesirable vegetation to achieve the maximum control of weeds.

By a “herbicide-tolerant” or “herbicide-resistant” plant, it is intended that a plant that is tolerant or resistant to at least one herbicide at a level that would normally kill, or inhibit the growth of, a normal or wildtype plant. Levels of herbicide that normally inhibit growth of a non-tolerant plant are known and readily determined by those skilled in the art. Examples include the amounts recommended by manufacturers for application. The maximum rate is an example of an amount of herbicide that would normally inhibit growth of a non-tolerant plant. For the present invention, the terms “herbicide-tolerant” and “herbicide-resistant” are used interchangeably and are intended to have an equivalent meaning and an equivalent scope. Similarly, the terms “herbicide-tolerance” and “herbicide-resistance” are used interchangeably and are intended to have an equivalent meaning and an equivalent scope. Similarly, the terms “tolerant” and “resistant” are used interchangeably and are intended to have an equivalent meaning and an equivalent scope. As used herein, in regard to an herbicidal composition useful in various embodiments hereof, terms such as herbicides, and the like, refer to those agronomically acceptable herbicide active ingredients (A. I.) recognized in the art. Similarly, terms such as fungicide, nematicide, pesticide, and the like, refer to other agronomically acceptable active ingredients recognized in the art.

When used in reference to a particular mutant enzyme or polypeptide, terms such as herbicide-tolerant and herbicide-tolerance refer to the ability of such enzyme or polypeptide to perform its physiological activity in the presence of an amount of an herbicide A.I. that would normally inactivate or inhibit the activity of the wild-type (non-mutant) version of said enzyme or polypeptide. Furthermore, the PPO activity of such a herbicide-tolerant or herbicide-resistant mutated PPO protein may be referred to herein as “herbicide-tolerant” or “herbicide-resistant” PPO activity.

As used herein, “recombinant,” when referring to nucleic acid or polypeptide, indicates that such material has been altered as a result of human application of a recombinant technique, such as by polynucleotide restriction and ligation, by polynucleotide overlap-extension, or by genomic insertion or transformation. A gene sequence open reading frame is recombinant if that nucleotide sequence has been removed from it natural text and cloned into any type of artificial nucleic acid vector. The term recombinant also can refer to an organism having a recombinant material, e.g., a plant that comprises a recombinant nucleic acid can be considered a recombinant plant.

The term “transgenic plant” refers to a plant that comprises a heterologous polynucleotide. Preferably, the heterologous polynucleotide is stably integrated within the genome such that the polynucleotide is passed on to successive generations. The heterologous polynucleotide may be integrated into the genome alone or as part of a recombinant expression cassette. “Transgenic” is used herein to refer to any cell, cell line, callus, tissue, plant part or plant, the genotype of which has been so altered by the presence of heterologous nucleic acid including those transgenic organisms or cells initially so altered, as well as those created by crosses or asexual propagation from the initial transgenic organism or cell. In some embodiments, a “recombinant” organism is a “transgenic” organism. The term “transgenic” as used herein is not intended to encompass the alteration of the genome (chromosomal or extra-chromosomal) by conventional plant breeding methods (e.g., crosses) or by naturally occurring events such as, e.g., self-fertilization, random cross-fertilization, non-recombinant viral infection, non-recombinant bacterial transformation, non-recombinant transposition, or spontaneous mutation.

As used herein, “mutagenized” refers to an organism or DNA thereof having alteration(s) in the biomolecular sequence of its native genetic material as compared to the sequence of the genetic material of a corresponding wild-type organism or DNA, wherein the alteration(s) in genetic material were induced and/or selected by human action. Examples of human action that can be used to produce a mutagenized organism or DNA include, but are not limited to, treatment with a chemical mutagen such as EMS and subsequent selection with herbicide(s); or by treatment of plant cells with x-rays and subsequent selection with herbicide(s). Any method known in the art can be used to induce mutations. Methods of inducing mutations can induce mutations in random positions in the genetic material or can induce mutations in specific locations in the genetic material (i.e., can be directed mutagenesis techniques), such as by use of a genoplasty technique.

As used herein, a “genetically modified organism” (GMO) is an organism whose genetic characteristics contain alteration(s) that were produced by human effort causing transfection that results in transformation of a target organism with genetic material from another or “source” organism, or with synthetic or modified-native genetic material, or an organism that is a descendant thereof that retains the inserted genetic material. The source organism can be of a different type of organism (e.g., a GMO plant can contain bacterial genetic material) or from the same type of organism (e.g., a GMO plant can contain genetic material from another plant). As used herein in regard to plants and other organisms, “recombinant,” “transgenic,” and “GMO” are considered synonyms and indicate the presence of genetic material from a different source; in contrast, “mutagenized” is used to refer to a plant or other organism, or the DNA thereof, in which no such transgenic material is present, but in which the native genetic material has become mutated so as to differ from a corresponding wild-type organism or DNA.

As used herein, “wild-type” or “corresponding wild-type plant” means the typical form of an organism or its genetic material, as it normally occurs, as distinguished from, e.g., mutagenized and/or recombinant forms. Similarly, by “control cell” or “similar, wild-type, plant, plant tissue, plant cell or host cell” is intended a plant, plant tissue, plant cell, or host cell, respectively, that lacks the herbicide-resistance characteristics and/or particular polynucleotide of the invention that are disclosed herein. The use of the term “wild-type” is not, therefore, intended to imply that a plant, plant tissue, plant cell, or other host cell lacks recombinant DNA in its genome, and/or does not possess herbicide-resistant characteristics that are different from those disclosed herein.

As used herein, “descendant” refers to any generation plant. In some embodiments, a descendant is a first, second, third, fourth, fifth, sixth, seventh, eight, ninth, or tenth generation plant.

As used herein, “progeny” refers to a first generation plant.

The term “seed” comprises seeds of all types, such as, for example, true seeds, caryopses, achenes, fruits, tubers, seedlings and similar forms. In the context of Brassica and Sinapis species, “seed” refers to true seed(s) unless otherwise specified. For example, the seed can be seed of transgenic plants or plants obtained by traditional breeding methods. Examples of traditional breeding methods can include cross-breeding, selfing, back-crossing, embryo rescue, in-crossing, out-crossing, inbreeding, selection, asexual propagation, and other traditional techniques as are known in the art.

Although exemplified with reference to specific plants or plant varieties and their hybrids, in various embodiments, the presently described methods using herbicides can be employed with a variety of commercially valuable plants. Herbicide-tolerant plant lines described as useful herein can be employed in weed control methods either directly or indirectly, i. e. either as crops for herbicide treatment or as herbicide-tolerance trait donor lines for development, to produce other varietal and/or hybrid crops containing such trait or traits. All such resulting variety or hybrids crops, containing the ancestral herbicide-tolerance trait or traits can be referred to herein as progeny or descendant of the ancestral, herbicide-tolerant line(s). Such resulting plants can be said to retain the “herbicide tolerance characteristic(s)” of the ancestral plant, i.e. meaning that they possess and express the ancestral genetic molecular components responsible for the trait.

In one aspect, the present invention provides a plant or plant part comprising a polynucleotide encoding a mutated PPO polypeptide, the expression of said polynucleotide confers to the plant or plant part tolerance to herbicides.

In a preferred embodiment, the plant has been previously produced by a process comprising recombinantly preparing a plant by introducing and over-expressing a mutated PPO transgene according to the present invention, as described in greater detail hereinafter.

In another embodiment, the polynucleotide encoding the mutated PPO polypeptide polypeptide comprises the nucleic acid sequence set forth in SEQ ID NO: 118, 119, 120, 121, 122, 123, 124, 125, 126, 127, 128, or 130, or a variant or derivative thereof.

In other embodiments, the mutated PPO polypeptide according to the present invention is a functional variant having, over the full-length of the variant, at least about 80%, illustratively, at least about 80%, 90%, 95%, 98%, 99% or more amino acid sequence identity to SEQ ID NO: 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, 100, 101, 102, 103, 104, 105, 106, 107, 108, 109, 110, 111, 112, 113, 114, 115, 116, 117, or 129.

In another embodiment, the mutated PPO polypeptide for use according to the present invention is a functional fragment of a polypeptide having the amino acid sequence set forth in SEQ ID NO: 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, 100, 101, 102, 103, 104, 105, 106, 107, 108, 109, 110, 111, 112, 113, 114, 115, 116, 117, or 129.

It is recognized that the PPO polynucleotide molecules and PPO polypeptides of the invention encompass polynucleotide molecules and polypeptides comprising a nucleotide or an amino acid sequence that is sufficiently identical to nucleotide sequence set forth in SEQ ID NOs: 118, 119, 120, 121, 122, 123, 124, 125, 126, 127, 128, or 130, or to the amino acid sequence set forth in SEQ ID Nos: 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, 100, 101, 102, 103, 104, 105, 106, 107, 108, 109, 110, 111, 112, 113, 114, 115, 116, 117, or 129. The term “sufficiently identical” is used herein to refer to a first amino acid or nucleotide sequence that contains a sufficient or minimum number of identical or equivalent (e.g., with a similar side chain) amino acid residues or nucleotides to a second amino acid or nucleotide sequence such that the first and second amino acid or nucleotide sequences have a common structural domain and/or common functional activity.

Generally, “sequence identity” refers to the extent to which two optimally aligned DNA or amino acid sequences are invariant throughout a window of alignment of components, e.g., nucleotides or amino acids. An “identity fraction” for aligned segments of a test sequence and a reference sequence is the number of identical components that are shared by the two aligned sequences divided by the total number of components in reference sequence segment, i.e., the entire reference sequence or a smaller defined part of the reference sequence. “Percent identity” is the identity fraction times 100. Optimal alignment of sequences for aligning a comparison window are well known to those skilled in the art and may be conducted by tools such as the local homology algorithm of Smith and Waterman, the homology alignment algorithm of Needleman and Wunsch, the search for similarity method of Pearson and Lipman, and preferably by computerized implementations of these algorithms such as GAP, BESTFIT, FASTA, and TFASTA available as part of the GCG.

Wisconsin Package. (Accelrys Inc. Burlington, Mass.)

Polynucleotides and Oligonucleotides

By an “isolated polynucleotide”, including DNA, RNA, or a combination of these, single or double stranded, in the sense or antisense orientation or a combination of both, dsRNA or otherwise, we mean a polynucleotide which is at least partially separated from the polynucleotide sequences with which it is associated or linked in its native state. Preferably, the isolated polynucleotide is at least 60% free, preferably at least 75% free, and most preferably at least 90% free from other components with which they are naturally associated. As the skilled addressee would be aware, an isolated polynucleotide can be an exogenous polynucleotide present in, for example, a transgenic organism which does not naturally comprise the polynucleotide. Furthermore, the terms “polynucleotide(s)”, “nucleic acid sequence(s)”, “nucleotide sequence(s)”, “nucleic acid(s)”, “nucleic acid molecule” are used interchangeably herein and refer to nucleotides, either ribonucleotides or deoxyribonucleotides or a combination of both, in a polymeric unbranched form of any length.

The term “mutated PPO nucleic acid” refers to a PPO nucleic acid having a sequence that is mutated from a wild-type PPO nucleic acid and that confers increased herbicide tolerance to a plant in which it is expressed. Furthermore, the term “mutated protoporphyrinogen oxidase (mutated PPO)” refers to the replacement of an amino acid of the wild-type primary sequence of SEQ ID NO: 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, 100, 101, 102, 103, 104, 105, 106, 107, 108, 109, 110, 111, 112, 113, 114, 115, 116, 117, or 129, or a variant, a derivative, a homologue, an orthologue, or paralogue thereof, with another amino acid. The expression “mutated amino acid” will be used below to designate the amino acid which is replaced by another amino acid, thereby designating the site of the mutation in the primary sequence of the protein.

In a preferred embodiment, the PPO nucleotide sequence encoding a mutated PPO comprises the sequence of SEQ ID NO: 118, 119, 120, 121, 122, 123, 124, 125, 126, 127, 128, or 130, or a variant or derivative thereof

Furthermore, it will be understood by the person skilled in the art that the PPO nucleotide sequences encompasse homologues, paralogues and and orthologues of SEQ ID NO: 118, 119, 120, 121, 122, 123, 124, 125, 126, 127, 128, or 130, as defined hereinafter.

The term “variant” with respect to a sequence (e.g., a polypeptide or nucleic acid sequence such as—for example—a transcription regulating nucleotide sequence of the invention) is intended to mean substantially similar sequences. For nucleotide sequences comprising an open reading frame, variants include those sequences that, because of the degeneracy of the genetic code, encode the identical amino acid sequence of the native protein. Naturally occurring allelic variants such as these can be identified with the use of well-known molecular biology techniques, as, for example, with polymerase chain reaction (PCR) and hybridization techniques. Variant nucleotide sequences also include synthetically derived nucleotide sequences, such as those generated, for example, by using site-directed mutagenesis and for open reading frames, encode the native protein comprising the sequence of SEQ ID NO: 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, 100, 101, 102, 103, 104, 105, 106, 107, 108, 109, 110, 111, 112, 113, 114, 115, 116, 117, or 129, as well as those that encode a polypeptide having amino acid substitutions relative to the native protein, e.g. the mutated PPO according to the present invention as disclosed herein. Generally, nucleotide sequence variants of the invention will have at least 30, 40, 50, 60, to 70%, e.g., preferably 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, to 79%, generally at least 80%, e.g., 81%-84%, at least 85%, e.g., 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, to 98% and 99% nucleotide “sequence identity” to the nucleotide sequence of SEQ ID NO: 118, 119, 120, 121, 122, 123, 124, 125, 126, 127, 128, or 130. The % identity of a polynucleotide is determined by GAP (Needleman and Wunsch, 1970) analysis (GCG program) with a gap creation penalty=5, and a gap extension penalty=0.3. Unless stated otherwise, the query sequence is at least 45 nucleotides in length, and the GAP analysis aligns the two sequences over a region of at least 45 nucleotides. Preferably, the query sequence is at least 150 nucleotides in length, and the GAP analysis aligns the two sequences over a region of at least 150 nucleotides. More preferably, the query sequence is at least 300 nucleotides in length and the GAP analysis aligns the two sequences over a region of at least 300 nucleotides. Even more preferably, the GAP analysis aligns the two sequences over their entire length.

Polypeptides

By “substantially purified polypeptide” or “purified” a polypeptide is meant that has been separated from one or more lipids, nucleic acids, other polypeptides, or other contaminating molecules with which it is associated in its native state. It is preferred that the substantially purified polypeptide is at least 60% free, more preferably at least 75% free, and more preferably at least 90% free from other components with which it is naturally associated. As the skilled addressee will appreciate, the purified polypeptide can be a recombinantly produced polypeptide. The terms “polypeptide” and “protein” are generally used interchangeably and refer to a single polypeptide chain which may or may not be modified by addition of non-amino acid groups. It would be understood that such polypeptide chains may associate with other polypeptides or proteins or other molecules such as co-factors. The terms “proteins” and “polypeptides” as used herein also include variants, mutants, modifications, analogous and/or derivatives of the polypeptides of the invention as described herein.

The % identity of a polypeptide is determined by GAP (Needleman and Wunsch, 1970) analysis (GCG program) with a gap creation penalty=5, and a gap extension penalty=0.3. The query sequence is at least 25 amino acids in length, and the GAP analysis aligns the two sequences over a region of at least 25 amino acids. More preferably, the query sequence is at least 50 amino acids in length, and the GAP analysis aligns the two sequences over a region of at least 50 amino acids. More preferably, the query sequence is at least 100 amino acids in length and the GAP analysis aligns the two sequences over a region of at least 100 amino acids. Even more preferably, the query sequence is at least 250 amino acids in length and the GAP analysis aligns the two sequences over a region of at least 250 amino acids. Even more preferably, the GAP analysis aligns the two sequences over their entire length.

With regard to a defined polypeptide, it will be appreciated that % identity figures higher than those provided above will encompass preferred embodiments. Thus, where applicable, in light of the minimum % identity figures, it is preferred that the PPO polypeptide of the invention comprises an amino acid sequence which is at least 40%, more preferably at least 45%, more preferably at least 50%, more preferably at least 55%, more preferably at least 60%, more preferably at least 65%, more preferably at least 70%, more preferably at least 75%, more preferably at least 80%, more preferably at least 85%, more preferably at least 90%, more preferably at least 91%, more preferably at least 92%, more preferably at least 93%, more preferably at least 94%, more preferably at least 95%, more preferably at least 96%, more preferably at least 97%, more preferably at least 98%, more preferably at least 99%, more preferably at least 99.1%, more preferably at least 99.2%, more preferably at least 99.3%, more preferably at least 99.4%, more preferably at least 99.5%, more preferably at least 99.6%, more preferably at least 99.7%, more preferably at least 99.8%, and even more preferably at least 99.9% identical to SEQ ID NO: 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, 100, 101, 102, 103, 104, 105, 106, 107, 108, 109, 110, 111, 112, 113, 114, 115, 116, 117, or 129.

By “variant” polypeptide is intended a polypeptide derived from the protein of SEQ ID NO: 2, by deletion (so-called truncation) or addition of one or more amino acids to the N-terminal and/or C-terminal end of the native protein; deletion or addition of one or more amino acids at one or more sites in the native protein; or substitution of one or more amino acids at one or more sites in the native protein. Such variants may result from, for example, genetic polymorphism or from human manipulation. Methods for such manipulations are generally known in the art.

“Derivatives” of a protein encompass peptides, oligopeptides, polypeptides, proteins and enzymes having amino acid substitutions, deletions and/or insertions relative to the unmodified protein in question and having similar biological and functional activity as the unmodified protein from which they are derived. Thus, functional variants and fragments of the PPO polypeptides, and nucleic acid molecules encoding them, also are within the scope of the present invention, and unless specifically described otherwise, irrespective of the origin of said polypeptide and irrespective of whether it occurs naturally. Various assays for functionality of a PPO polypeptide can be employed. For example, a functional variant or fragment of the PPO polypeptide can be assayed to determine its ability to confer herbicides detoxification. By way of illustration, a herbicides detoxification rate can be defined as a catalytic rate sufficient to provide a determinable increase in tolerance to herbicides in a plant or plant part comprising a recombinant polynucleotide encoding the variant or fragment of the PPO polypeptide, wherein the plant or plant part expresses the variant or fragment at up to about 0.5%, illustratively, about 0.05 to about 0.5%, about 0.1 to about 0.4%, and about 0.2 to about 0.3%, of the total cellular protein relative to a similarly treated control plant that does not express the variant or fragment.

In a preferred embodiment, the mutated PPO polypeptide is a functional variant or fragment of a protoporphyrinogen oxidase having the amino acid sequence set forth in SEQ ID NO: 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, 100, 101, 102, 103, 104, 105, 106, 107, 108, 109, 110, 111, 112, 113, 114, 115, 116, 117, or 129, wherein the functional variant or fragment has at least about 80% amino acid sequence identity to SEQ ID NO: 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, 100, 101, 102, 103, 104, 105, 106, 107, 108, 109, 110, 111, 112, 113, 114, 115, 116, 117, or 129.

In other embodiments, the functional variant or fragment further has a herbicides detoxification rate defined as a catalytic rate sufficient to provide a determinable increase in tolerance to herbicides in a plant or plant part comprising a recombinant polynucleotide encoding the variant or fragment, wherein the plant or plant part expresses the variant or fragment at up to about 0.5% of the total cellular protein to a similarly treated control plant that does not express the variant or fragment.

“Homologues” of a protein encompass peptides, oligopeptides, polypeptides, proteins and enzymes having amino acid substitutions, deletions and/or insertions relative to the unmodified protein in question and having similar biological and functional activity as the unmodified protein from which they are derived.

In addition, one of ordinary skill in the art will further appreciate that changes can be introduced by mutation into the nucleotide sequences of the invention thereby leading to changes in the amino acid sequence of the encoded proteins without altering the biological activity of the proteins.

Thus, for example, an isolated polynucleotide molecule encoding a mutated PPO polypeptide having an amino acid sequence that differs from that of SEQ ID NO: 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, 100, 101, 102, 103, 104, 105, 106, 107, 108, 109, 110, 111, 112, 113, 114, 115, 116, 117, or 129 can be created by introducing one or more nucleotide substitutions, additions, or deletions into the corresponding nucleotide sequence, such that one or more amino acid substitutions, additions or deletions are introduced into the encoded protein. Mutations can be introduced by standard techniques, such as site-directed mutagenesis and PCR-mediated mutagenesis. Such variant nucleotide sequences are also encompassed by the present invention. For example, preferably, conservative amino acid substitutions may be made at one or more predicted preferably nonessential amino acid residues. A “nonessential” amino acid residue is a residue that can be altered from the wild-type sequence of a protein without altering the biological activity, whereas an “essential” amino acid residue is required for biological activity.

A deletion refers to removal of one or more amino acids from a protein.

An insertion refers to one or more amino acid residues being introduced into a predetermined site in a protein. Insertions may comprise N-terminal and/or C-terminal fusions as well as intra-sequence insertions of single or multiple amino acids. Generally, insertions within the amino acid sequence will be smaller than N- or C-terminal fusions, of the order of about 1 to 10 residues. Examples of N- or C-terminal fusion proteins or peptides include the binding domain or activation domain of a transcriptional activator as used in the yeast two-hybrid system, phage coat proteins, (histidine)-6-tag, glutathione S-transferase-tag, protein A, maltose-binding protein, dihydrofolate reductase, Tag⋅100 epitope, c-myc epitope, FLAG®-epitope, lacZ, CMP (calmodulin-binding peptide), HA epitope, protein C epitope and VSV epitope.

A substitution refers to replacement of amino acids of the protein with other amino acids having similar properties (such as similar hydrophobicity, hydrophilicity, antigenicity, propensity to form or break a-helical structures or 13-sheet structures). Amino acid substitutions are typically of single residues, but may be clustered depending upon functional constraints placed upon the polypeptide and may range from 1 to 10 amino acids; insertions will usually be of the order of about 1 to 10 amino acid residues. A conservative amino acid substitution is one in which the amino acid residue is replaced with an amino acid residue having a similar side chain. Families of amino acid residues having similar side chains have been defined in the art. These families include amino acids with basic side chains (e.g., lysine, arginine, histidine), acidic side chains (e.g., aspartic acid, glutamic acid), uncharged polar side chains (e.g., glycine, asparagine, glutamine, serine, threonine, tyrosine, cysteine), nonpolar side chains (e.g., alanine, valine, leucine, isoleucine, proline, phenylalanine, methionine, tryptophan), beta-branched side chains (e.g., threonine, valine, isoleucine) and aromatic side chains (e.g., tyrosine, phenylalanine, tryptophan, histidine). Such substitutions would not be made for conserved amino acid residues, or for amino acid residues residing within a conserved motif. Conservative substitution tables are well known in the art (see for example Creighton (1984) Proteins. W.H. Freeman and Company (Eds).

Amino acid substitutions, deletions and/or insertions may readily be made using peptide synthetic techniques well known in the art, such as solid phase peptide synthesis and the like, or by recombinant DNA manipulation. Methods for the manipulation of DNA sequences to produce substitution, insertion or deletion variants of a protein are well known in the art. For example, techniques for making substitution mutations at predetermined sites in DNA are well known to those skilled in the art and include M13 mutagenesis, T7-Gen in vitro mutagenesis (USB, Cleveland, Ohio), QuickChange Site Directed mutagenesis (Stratagene, San Diego, Calif.), PCR-mediated site-directed mutagenesis or other site-directed mutagenesis protocols.

“Derivatives” further include peptides, oligopeptides, polypeptides which may, compared to the amino acid sequence of the naturally-occurring form of the protein, such as the protein of interest, comprise substitutions of amino acids with non-naturally occurring amino acid residues, or additions of non-naturally occurring amino acid residues. “Derivatives” of a protein also encompass peptides, oligopeptides, polypeptides which comprise naturally occurring altered (glycosylated, acylated, prenylated, phosphorylated, myristoylated, sulphated etc.) or non-naturally altered amino acid residues compared to the amino acid sequence of a naturally-occurring form of the polypeptide. A derivative may also comprise one or more non-amino acid substituents or additions compared to the amino acid sequence from which it is derived, for example a reporter molecule or other ligand, covalently or non-covalently bound to the amino acid sequence, such as a reporter molecule which is bound to facilitate its detection, and non-naturally occurring amino acid residues relative to the amino acid sequence of a naturally-occurring protein. Furthermore, “derivatives” also include fusions of the naturally-occurring form of the protein with tagging peptides such as FLAG, HIS6 or thioredoxin (for a review of tagging peptides, see Terpe, Appl. Microbiol. Biotechnol. 60, 523-533, 2003).

“Orthologues” and “paralogues” encompass evolutionary concepts used to describe the ancestral relationships of genes. Paralogues are genes within the same species that have originated through duplication of an ancestral gene; orthologues are genes from different organisms that have originated through speciation, and are also derived from a common ancestral gene.

It is well-known in the art that paralogues and orthologues may share distinct domains harboring suitable amino acid residues at given sites, such as binding pockets for particular substrates or binding motifs for interaction with other proteins.

The term “domain” refers to a set of amino acids conserved at specific positions along an alignment of sequences of evolutionarily related proteins. While amino acids at other positions can vary between homologues, amino acids that are highly conserved at specific positions indicate amino acids that are likely essential in the structure, stability or function of a protein. Identified by their high degree of conservation in aligned sequences of a family of protein homologues, they can be used as identifiers to determine if any polypeptide in question belongs to a previously identified polypeptide family.

The term “motif” or “consensus sequence” refers to a short conserved region in the sequence of evolutionarily related proteins. Motifs are frequently highly conserved parts of domains, but may also include only part of the domain, or be located outside of conserved domain (if all of the amino acids of the motif fall outside of a defined domain).

Specialist databases exist for the identification of domains, for example, SMART (Schultz et al. (1998) Proc. Natl. Acad. Sci. USA 95, 5857-5864; Letunic et al. (2002) Nucleic Acids Res 30, 242-244), InterPro (Mulder et al., (2003) Nucl. Acids. Res. 31, 315-318), Prosite (Bucher and Bairoch (1994), A generalized profile syntax for biomolecular sequences motifs and its function in automatic sequence interpretation. (In) ISMB-94; Proceedings 2nd International Conference on Intelligent Systems for Molecular Biology. Altman R., Brutlag D., Karp P., Lathrop R., Searls D., Eds., pp 53-61, AAAI Press, Menlo Park; Hulo et al., Nucl. Acids. Res. 32:D134-D137, (2004)), or Pfam (Bateman et al., Nucleic Acids Research 30(1): 276-280 (2002)). A set of tools for in silico analysis of protein sequences is available on the ExPASy proteomics server (Swiss Institute of Bioinformatics (Gasteiger et al., ExPASy: the proteomics server for in-depth protein knowledge and analysis, Nucleic Acids Res. 31:3784-3788(2003)). Domains or motifs may also be identified using routine techniques, such as by sequence alignment.

Methods for the alignment of sequences for comparison are well known in the art, such methods include GAP, BESTFIT, BLAST, FASTA and TFASTA. GAP uses the algorithm of Needleman and Wunsch ((1970) J Mol Biol 48: 443-453) to find the global (i.e. spanning the complete sequences) alignment of two sequences that maximizes the number of matches and minimizes the number of gaps. The BLAST algorithm (Altschul et al. (1990) J Mol Biol 215: 403-10) calculates percent sequence identity and performs a statistical analysis of the similarity between the two sequences. The software for performing BLAST analysis is publicly available through the National Centre for Biotechnology Information (NCBI). Homologues may readily be identified using, for example, the ClustalW multiple sequence alignment algorithm (version 1.83), with the default pairwise alignment parameters, and a scoring method in percentage. Global percentages of similarity and identity may also be determined using one of the methods available in the MatGAT software package (Campanella et al., BMC Bioinformatics. 2003 Jul. 10; 4:29. MatGAT: an application that generates similarity/identity matrices using protein or DNA sequences.). Minor manual editing may be performed to optimise alignment between conserved motifs, as would be apparent to a person skilled in the art. Furthermore, instead of using full-length sequences for the identification of homologues, specific domains may also be used. The sequence identity values may be determined over the entire nucleic acid or amino acid sequence or over selected domains or conserved motif(s), using the programs mentioned above using the default parameters. For local alignments, the Smith-Waterman algorithm is particularly useful (Smith T F, Waterman M S (1981) J. Mol. Biol 147(1);195-7).

The proteins of the invention may be altered in various ways including amino acid substitutions, deletions, truncations, and insertions. Methods for such manipulations are generally known in the art. For example, amino acid sequence variants can be prepared by mutations in the DNA. Methods for mutagenesis and nucleotide sequence alterations are well known in the art. See, for example, Kunkel (1985) PNAS, 82:488-492; Kunkel et al. (1987) Methods in Enzymol. 154:367-382; U.S. Pat. No. 4,873,192; Walker and Gaastra, eds. (1983) Techniques in Molecular Biology (MacMillan Publishing Company, New York) and the references cited therein. Guidance as to appropriate amino acid substitutions that do not affect biological activity of the protein of interest may be found in the model of Dayhoff et al. (1978) Atlas of Protein Sequence and Structure (Natl. Biomed. Res. Found., Washington, D. C), herein incorporated by reference. Conservative substitutions, such as exchanging one amino acid with another having similar properties, may be preferable.

Alternatively, variant nucleotide sequences can be made by introducing mutations randomly along all or part of a coding sequence, such as by saturation mutagenesis, and the resultant mutants can be screened to identify mutants that encode proteins that retain activity. For example, following mutagenesis, the encoded protein can be expressed recombinantly, and the activity of the protein can be determined using standard assay techniques.

The inventors of the present invention have found that by substituting one or more of the amino acid residues in the non-actives sites of the PPO enzyme of SEQ ID NO: 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, 100, 101, 102, 103, 104, 105, 106, 107, 108, 109, 110, 111, 112, 113, 114, 115, 116, 117, or 129, e.g. by employing one of the above described methods to mutate the PPO encoding nucleic acids, the tolerance or resistance to particular herbicides could be remarkably increased.

Preferred substitutions of mutated PPO are those that increase the herbicide tolerance of the plant, but leave the biological activity of the PPO enzymatic activity substantially unaffected.

Accordingly, in another object of the present invention refers to a mutated PPO polypeptide, comprising the sequence of SEQ ID NO: 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, 100, 101, 102, 103, 104, 105, 106, 107, 108, 109, 110, 111, 112, 113, 114, 115, 116, 117, or 129, a variant, derivative, orthologue, paralogue or homologue thereof, the key amino acid residues of which is substituted by any other amino acid.

It will be understood by the person skilled in the art that amino acids located in a close proximity to the positions of amino acids mentioned below may also be substituted. Thus, in another embodiment the variant of SEQ ID NO: 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, 100, 101, 102, 103, 104, 105, 106, 107, 108, 109, 110, 111, 112, 113, 114, 115, 116, 117, or 129, a variant, derivative, orthologue, paralogue or homologue thereof comprises a mutated PPO, wherein an amino acid ±3, ±2 or ±1 amino acid positions from a key amino acid is substituted by any other amino acid.

Based on techniques well-known in the art, a highly characteristic sequence pattern can be developed, by means of which further of mutated PPO candidates with the desired activity may be searched.

Searching for further mutated PPO candidates by applying a suitable sequence pattern would also be encompassed by the present invention. It will be understood by a skilled reader that the present sequence pattern is not limited by the exact distances between two adjacent amino acid residues of said pattern. Each of the distances between two neighbours in the above patterns may, for example, vary independently of each other by up to ±10, ±5, ±3, ±2 or ±1 amino acid positions without substantially affecting the desired activity. Furthermore, by applying the method of site directed mutagenesis, e.g. saturation mutagenes (see e.g. Schenk et al., Biospektrum 03/2006, pages 277-279), the inventors of the present invention have identified and generated specific amino acid substitutions and combinations thereof, which—when introduced into a plant by transforming and expressing the respective mutated PPO encoding nucleic acid—confer increased herbicide resistance or tolerance to a herbicide to said plant.

Thus, in a particularly preferred embodiment, the variant or derivative of the mutated PPO refers to a PPO polypeptide comprising SEQ ID NO: 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, 100, 101, 102, 103, 104, 105, 106, 107, 108, 109, 110, 111, 112, 113, 114, 115, 116, 117, or 129, a orthologue, paralogue, or homologue thereof, wherein the amino acid sequence differs from the wildtype amino acid sequence of a PPO polypeptide at one or more positions corresponding to the following positions of SEQ ID NO: 1 or 2: 32, 53, 57, 61, 63, 64, 65, 67, 71, 76, 82, 83, 85, 86, 87, 88, 91, 103, 104, 106, 108, 116, 119, 126, 127, 129, 139, 159, 210, 211, 224, 245, 246, 248, 249, 252, 253, 254, 255, 257, 259, 260, 262, 264, 286, 291, 305, 308, 309, 323, 335, 343, 345, 358, 372, 373, 387, 391, 392, 400, 412, 414, 415, 425, 428, 431, 433, 434, 435, 436, 447, 451, 453, 464, 466, 482 of SEQ ID NO: 1 or 2, wherein said difference refers to a substitution of the amino acid at that positions by any other amino acid.

In a particularly preferred embodiment, the variant or derivative of the mutated PPO refers to a PPO polypeptide comprising SEQ ID NO: 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, 100, 101, 102, 103, 104, 105, 106, 107, 108, 109, 110, 111, 112, 113, 114, 115, 116, 117, or 129, a orthologue, paralogue, or homologue thereof, wherein the amino acid sequence differs from the wildtype amino acid sequence of a PPO polypeptide at one or more positions corresponding to the following positions of SEQ ID NO: 1 or 2: 40, 250, 391, 520, 521, 522, 523, 524, 525, 526, 527, 528, 529, 530, 531, 532, 533, 534 of SEQ ID NO: 1 or 2, wherein said difference refers to a deletion or an insertion of the amino acid at that positions.

Preferably, the difference corresponding to position 391 of SEQ ID NO:1 refers to an insertion, and the difference corresponding to positions 40, 250, 520, 521, 522, 523, 524, 525, 526, 527, 528, 529, 530, 531, 532, 533, 534 of SEQ ID NO: 1 or 2 refers to a deletion

Preferably, said differences at these amino acid positions (corresponding to the respective positions in SEQ ID NO: 2 or 4) include, but are not limited to, one or more of the following:

the amino acid corresponding to position G32 is substituted by any other amino acid

the amino acid corresponding to position V53 is substituted by any other amino acid

the amino acid corresponding to position A57 is substituted by any other amino acid

the amino acid corresponding to position K61 is substituted by any other amino acid

the amino acid corresponding to position K63 is substituted by any other amino acid

the amino acid corresponding to position S64 is substituted by any other amino acid

the amino acid corresponding to position H65 is substituted by any other amino acid

the amino acid corresponding to position L67 is substituted by any other amino acid

the amino acid corresponding to position L71 is substituted by any other amino acid

the amino acid corresponding to position S76 is substituted by any other amino acid

the amino acid corresponding to position L82 is substituted by any other amino acid

the amino acid corresponding to position K83 is substituted by any other amino acid

the amino acid corresponding to position V85 is substituted by any other amino acid

the amino acid corresponding to position K86 is substituted by any other amino acid

the amino acid corresponding to position K87 is substituted by any other amino acid

the amino acid corresponding to position D88 is substituted by any other amino acid

the amino acid corresponding to position I91 is substituted by any other amino acid

the amino acid corresponding to position E103 is substituted by any other amino acid

the amino acid corresponding to position A104 is substituted by any other amino acid

the amino acid corresponding to position V106 is substituted by any other amino acid

the amino acid corresponding to position S108 is substituted by any other amino acid

the amino acid corresponding to position R116 is substituted by any other amino acid

the amino acid corresponding to position Q119 is substituted by any other amino acid,

preferably Leu

the amino acid corresponding to position K127 is substituted by any other amino acid

the amino acid corresponding to position N126 is substituted by any other amino acid

the amino acid corresponding to position Y129 is substituted by any other amino acid

the amino acid corresponding to position L139 is substituted by any other amino acid

the amino acid corresponding to position Q159 is substituted by any other amino acid

the amino acid corresponding to position G210 is substituted by any other amino acid,

preferably by Ser;

the amino acid corresponding to position G211 is substituted by any other amino acid,

preferably by Asp, Asn, Thr or Ala;

the amino acid corresponding to position E224 is substituted by any other amino acid

the amino acid corresponding to position L245 is substituted by any other amino acid

the amino acid corresponding to position K248 is substituted by any other amino acid

the amino acid corresponding to position S246 is substituted by any other amino acid

the amino acid corresponding to position E249 is substituted by any other amino acid

the amino acid corresponding to position G252 is substituted by any other amino acid

the amino acid corresponding to position E253 is substituted by any other amino acid

the amino acid corresponding to position N254 is substituted by any other amino acid

the amino acid corresponding to position A255 is substituted by any other amino acid

the amino acid corresponding to position I257 is substituted by any other amino acid

the amino acid corresponding to position K259 is substituted by any other amino acid

the amino acid corresponding to position P260 is substituted by any other amino acid

the amino acid corresponding to position V262 is substituted by any other amino acid

the amino acid corresponding to position G264 is substituted by any other amino acid

the amino acid corresponding to position D286 is substituted by any other amino acid

the amino acid corresponding to position Q291 is substituted by any other amino acid

the amino acid corresponding to position P305 is substituted by any other amino acid

the amino acid corresponding to position G308 is substituted by any other amino acid

the amino acid corresponding to position N309 is substituted by any other amino acid

the amino acid corresponding to position Q323 is substituted by any other amino acid

the amino acid corresponding to position R335 is substituted by any other amino acid

the amino acid corresponding to position M343 is substituted by any other amino acid

the amino acid corresponding to position F345 is substituted by any other amino acid

the amino acid corresponding to position T358 is substituted by any other amino acid

the amino acid corresponding to position D372 is substituted by any other amino acid

the amino acid corresponding to position K373 is substituted by any other amino acid

the amino acid corresponding to position S387 is substituted by any other amino acid,

preferably Leu

the amino acid corresponding to position H391 is substituted by any other amino acid

the amino acid corresponding to position N392 is substituted by any other amino acid

the amino acid corresponding to position L400 is substituted by any other amino acid

the amino acid corresponding to position S412 is substituted by any other amino acid,

preferably by Asn;

the amino acid corresponding to position M414 is substituted by any other amino acid

the amino acid corresponding to position C415 is substituted by any other amino acid

the amino acid corresponding to position R425 is substituted by any other amino acid

the amino acid corresponding to position K428 is substituted by any other amino acid

the amino acid corresponding to position N431 is substituted by any other amino acid

the amino acid corresponding to position S433 is substituted by any other amino acid

the amino acid corresponding to position M434 is substituted by any other amino acid

the amino acid corresponding to position D435 is substituted by any other amino acid

the amino acid corresponding to position E436 is substituted by any other amino acid

the amino acid corresponding to position Q447 is substituted by any other amino acid

the amino acid corresponding to position T451 is substituted by any other amino acid

the amino acid corresponding to position D453 is substituted by any other amino acid

the amino acid corresponding to position S464 is substituted by any other amino acid

the amino acid corresponding to position Q466 is substituted by any other amino acid

the amino acid corresponding to position D482 is substituted by any other amino acid

The corresponding residues in other species are shown in the following table:

SEQ ID Pos 1 Pos 2 Pos 3 Pos 4 Pos 5 Pos 6 Pos 7 Pos 8 Pos 9 Pos 10  1 G32 V53 A57 K61 K63 S64 H65 L67 L71 S76  2 G32 V53 A57 K61 K63 S64 H65 L67 L71 S76  3 G32 V53 A57 K61 K63 S64 H65 L67 L71 S76  4 G32 V53 A57 K61 K63 S64 H65 L67 L71 S76  5 G32 V53 A57 K61 K63 S64 H65 L67 L71 S76  6 G2 V23 A27 K31 K33 S34 H35 L37 L41 S46  7 V30 V52 A56 K60 K62 S63 N64 L66 L70 S75  8 K4 V25 A29 K33 K35 L36 H37 V39 L43 E48  9 S2 V24 A28 K32 K34 S35 H36 L38 V42 E47  10 N6 V23 A27 K31 K33 S34 H35 L37 M41 G46  11 T6 V23 A27 K31 K33 S34 H35 L37 V41 G46  12 L4 V21 A25 K29 K31 S32 H33 F35 V39 G44  13 K6 V23 A27 K31 K33 S34 H35 L37 V41 G46  14 Q6 V23 A27 K31 K33 S34 H35 F37 V41 G46  15 G6 V23 A27 K31 K33 S34 N35 V37 V41 E46  16 G11 V23 A27 K31 K33 S34 N35 V37 V41 E46  17 K6 V23 A27 K31 K33 S34 H35 L37 V41 G46  18 A5 V23 A27 K31 K33 S34 H35 F37 V41 E46  19 — V18 A22 K26 K28 S29 H30 F32 V36 E41  20 G6 V23 A27 K31 K33 I34 H35 L37 V41 G46  21 G6 V20 A24 K28 K30 V31 H32 L34 V38 G43  22 G4 V26 A30 K34 K36 S37 R38 L40 V44 G49  23 G7 V28 A32 K36 K38 S39 R40 L42 V46 E51  24 G4 V26 A30 K34 K36 S37 R38 L40 V44 G49  25 G50 V72 A76 K80 K82 S83 R84 L86 V90 G95  26 — — A3 K7 K9 L10 H11 L13 V17 E22  27 G34 V53 A57 R61 R63 K64 S65 V67 V71 D76  28 G36 V55 V59 R63 R65 K66 S67 V69 V73 D78  29 G4 V26 A30 K34 K36 S37 R38 L40 V44 G49  30 G36 V55 A59 R63 R65 Q66 S67 V69 V73 D78  31 G36 V55 A59 R63 R65 Q66 S67 V69 V73 D78  32 G8 I31 A35 K39 K41 S42 N43 F45 V49 G54  33 A41 I69 Q73 A77 R86 S87 S88 P90 V94 D99  34 A4 V25 A29 K33 K35 S36 N37 V39 V43 G48  35 A69 V90 A94 K98 K100 S101 K102 L104 V108 G113  36 L36 V59 V63 R67 R69 K70 S71 V73 V77 D82  37 — V20 A24 R28 R30 K31 R32 V34 V38 D43  38 H4 I25 A29 R33 K35 S36 Q37 L39 I43 G48  39 A2 V23 A27 K31 K33 I34 H35 L37 V41 G46  40 — V20 A24 R28 R30 K31 R32 V34 V38 D43  41 — V20 A24 R28 R30 K31 R32 V34 V38 D43  42 A7 V28 — R29 K31 S32 R33 L35 V39 G44  43 M1 A22 A26 R30 R32 A33 A34 V36 V40 N45  44 M1 A22 A26 R30 R32 A33 A34 V36 V40 N45  45 G32 V53 A57 K61 K63 S64 H65 L67 L71 S76  46 G32 V53 A57 K61 K63 S64 H65 L67 L71 S76  47 — — — — — — — — — —  48 — — — — — — — — — —  49 — V14 S18 K22 K24 L25 N26 L28 V32 G37  50 — — — — — — — — — —  51 — I11 T15 E19 H21 R22 R23 L25 V29 D34  52 — I11 A15 R19 Q21 E22 H23 H25 L29 G34  53 — I11 A15 R19 Q21 E22 H23 H25 V29 G34  54 — — — — — — — — — —  55 — I11 A15 E19 H21 R22 R23 L25 V29 D34  56 — I10 T14 Y18 K20 Q21 Q22 V24 V28 N33  57 S4 I27 T31 R35 T37 Q38 L39 H41 V45 D50  58 — I15 T19 E23 L25 K26 L27 H29 V33 S38  59 — I12 T16 L20 S22 K23 R24 F26 130 S35  60 — I12 Q16 E20 K22 K23 S24 F26 V30 E35  61 — I11 T15 Q19 Q21 Q22 Q23 H25 V29 G34  62 A2 V23 A27 K31 K33 L34 H35 L37 V41 E46  63 — I12 S16 W20 V22 K23 K24 Y26 I30 N35  64 M1 I22 A26 H30 S32 R33 M34 K36 V40 S45  65 — P16 S20 R24 L26 H27 P31 L33 L39 G44  66 — V12 S16 H20 Q22 Q23 A24 H26 V30 K35  67 — I12 S16 F20 K22 R23 A24 A26 V30 E35  68 — I11 A15 Q19 Q21 R22 K23 K25 V29 P34  69 — I13 S17 T21 F23 K24 R25 L27 V31 A36  70 — I10 T14 R18 A20 R21 E22 H24 L28 P33  71 — V12 T16 R20 K22 S23 H24 L26 L30 G35  72 P8 I29 T33 R37 A39 R40 E41 H43 L47 P52  73 — I14 A18 T22 H24 K25 R26 Y28 V32 N37  74 — V15 A19 T23 K25 N26 R27 V29 L33 G38  75 — V15 A19 T23 K25 N26 R27 V29 L33 G38  76 — L13 S17 S21 E23 N24 K25 N27 I31 L36  77 — I15 A19 T23 Y25 K26 R27 Y29 V33 N38  78 — I10 S14 Y18 K20 K21 G22 A24 V28 K33  79 — V11 S15 Y19 E21 K22 F23 G25 I32 N37  80 — I14 S18 S22 N24 R25 S28 R30 V37 N42  81 — I15 A19 T23 K25 N26 R27 V29 L33 G38  82 — I11 T15 L19 Q21 K22 Q23 Q25 V29 K34  83 — L13 S17 S21 E23 N24 K25 N27 I31 L36  84 — I20 T24 R28 Q30 Q31 R36 G38 L45 S50  85 — I10 V14 Q18 Q20 K21 A22 V24 L28 T33  86 — I10 S14 Y18 Q20 R21 Q22 V24 L28 S33  87 — I15 A19 H23 K25 L26 A27 R29 L33 D38  88 — L14 T18 H22 R24 H25 K26 Q28 V32 E37  89 — I9 S13 H17 Q19 K20 L21 K23 L27 D32  90 — I11 A15 E19 R21 N22 I29 L31 L35 E40  91 — I12 T16 R20 Q22 Q23 S31 Q33 L37 S42  92 — I11 S15 A19 H21 Q22 D23 R25 L29 E34  93 — I17 S21 H25 Q27 E28 Q35 V37 L41 P46  94 — I11 A15 L19 R21 E22 M29 L31 I35 D40  95 — I11 A15 F19 R21 K22 Q23 W25 L29 A34  96 — L13 T17 Y21 R23 R24 K25 Q27 V31 D36  97 — V10 S14 Y18 K20 R21 G22 A24 L28 N33  98 G41 I65 C69 A73 V75 T76 A81 K83 V87 D92  99 N4 V25 A29 K33 K35 S36 K37 V39 V43 G48 100 S47 I68 C72 A76 A78 T79 V84 S86 V90 D95 101 A2 V23 A27 K31 K33 S34 Q35 L37 V41 G46 102 A45 I67 C71 A75 S77 T78 H80 G82 V86 E91 103 T6 V27 A31 K35 K37 L38 H39 I41 V45 E50 104 — I94 C98 A102 S104 T105 L110 T112 V116 D121 105 S36 I60 C64 A68 A70 T71 G74 T76 V80 A85 106 M1 V22 V26 R30 R32 K33 S34 V36 V40 D45 107 S47 I68 C72 A76 A78 T79 V84 S86 V90 D95 108 A64 I89 C93 A97 C99 T100 L105 P107 V111 D116 109 T38 I59 C63 A67 A69 T70 G73 G75 V79 D84 110 L47 V67 V71 M75 R77 K78 S79 V81 V85 D90 111 T39 I60 C64 A68 A70 T71 G74 S76 V80 D85 112 — V20 A24 K28 K30 V31 H32 L34 V38 G43 113 T54 I75 C79 A83 A85 T86 G89 S91 V95 D100 114 S47 I68 C72 A76 A78 T79 V84 S86 V90 D95 115 A2 V23 A27 K31 K33 S34 Q35 L37 V41 G46 116 A64 I89 C93 A97 C99 T100 L105 P107 V111 D116 117 S3 V24 A28 K32 K34 S35 K36 V38 V42 G47 129 Y32 I62 V66 R70 S72 K73 S74 V76 V80 D85 SEQ ID Pos 11 Pos 12 Pos 13 Pos 14 Pos 15 Pos 16 Pos 17 Pos 18 Pos 19 Pos 20  1 L82 K83 V85 K86 K87 D88 I91 E103 A104 V106  2 L82 K83 V85 K86 K87 D88 I91 E103 A104 V106  3 L82 K83 V85 K86 K87 D88 I91 E103 A104 V106  4 L82 K83 V85 K86 K87 D88 I91 E103 A104 V106  5 L82 K83 V85 K86 K87 D88 I91 E103 A104 V106  6 L52 K53 V55 K56 K57 D58 I61 E73 A74 V76  7 L81 K82 V84 V85 K86 D87 I90 D102 E103 V105  8 L54 R55 V57 S58 Q59 H60 V63 E75 I76 V78  9 L53 R54 V56 N57 H58 D59 I62 E74 M75 V77  10 L52 R53 V55 S56 Q57 E58 I61 E73 I74 V76  11 L52 R53 V55 S56 Q57 D58 I61 E73 I74 V76  12 L50 R51 V53 S54 H55 D56 I59 E71 K72 V74  13 L52 R53 V55 S56 R57 D58 V61 E73 I74 V76  14 L52 R53 V55 S56 Y57 N58 I61 E73 T74 V76  15 L52 R53 I55 S56 K57 D58 I61 E73 M74 V76  16 L52 R53 I55 S56 K57 D58 I61 E73 M74 V76  17 L52 R53 V55 S56 R57 D58 V61 E73 A74 V76  18 L52 R53 V55 S56 Y57 K58 I61 E73 P74 V76  19 L47 R48 V50 S51 Y52 K53 I56 E68 P69 V71  20 L52 R53 V55 S56 Q57 D58 I61 E73 G74 V76  21 L49 R50 L52 S53 Q54 D55 I58 E70 G71 V73  22 L55 R56 V58 M59 Q60 N61 I64 E76 P77 V79  23 L57 T58 V60 M61 Q62 N63 I66 E78 P79 V81  24 L55 R56 V58 M59 Q60 N61 I64 E76 P77 V79  25 L101 R102 V104 M105 Q106 N107 I110 E122 P123 V125  26 L28 R29 V31 S32 Q33 D34 I37 E49 A50 V52  27 I82 R83 N85 S86 E87 A88 L91 E103 L104 V106  28 I84 R85 N87 S88 E89 G90 L93 E105 L106 A108  29 L55 R56 V58 M59 Q60 N61 I64 E76 P77 V79  30 I84 R85 N87 S88 E89 G90 V93 E105 W106 A108  31 I84 R85 N87 S88 E89 G90 V93 E105 W106 A108  32 I60 R61 G63 S64 Q65 E66 I69 — V81 A83  33 I105 T106 V108 E109 R110 D111 L114 D126 A127 L129  34 L54 R55 V57 S58 H59 H60 V63 E75 V76 V78  35 L119 R120 V122 M123 H124 N125 I128 E140 P141 V143  36 I88 R89 N91 S92 D93 S94 L97 A109 L110 A112  37 I49 R50 N52 S53 E54 G55 I58 E70 L71 A73  38 I54 K55 F57 A58 Q59 N60 I63 E75 P76 V78  39 L52 R53 L55 S56 Q57 D58 I61 E73 G74 V76  40 I49 R50 N52 S53 E54 G55 I58 E70 L71 A73  41 I49 R50 N52 S53 E54 G55 I58 E70 L71 A73  42 L50 R51 V53 M54 H55 K56 I59 E71 P72 V74  43 L51 K52 V54 S55 E56 N57 I60 D72 P73 I75  44 L51 K52 V54 S55 E56 N57 I60 D72 P73 I75  45 L82 K83 V85 K86 K87 D88 I91 E103 A104 V106  46 L82 K83 V85 K86 K87 D88 I91 E103 A104 V106  47 — — — — — — — — — —  48 — — — — — — — A5 L6 A8  49 I43 R44 S46 S47 Q48 D49 I52 E64 E65 V67  50 — — — — — — — — — —  51 I40 Q41 E43 R44 I45 D46 L49 S61 G62 F64  52 I40 R41 E43 S44 S45 E46 L49 A61 A62 L64  53 I40 R41 E43 S44 S45 E46 L49 A61 A62 L64  54 — — — — — — — — — —  55 I40 Q41 E43 R44 I45 D46 L49 S61 G62 F64  56 V39 I40 E42 K43 K44 D45 L48 S60 Q61 L63  57 I56 K57 E59 E60 V61 D62 L65 E77 L78 V80  58 I44 E45 I47 R48 Q49 N50 L53 S65 P66 I68  59 I41 E42 I44 K45 E46 N47 L50 T62 P63 I65  60 I41 G42 T44 A45 R46 D47 R50 S62 Q63 T65  61 I40 R41 E43 R44 T45 D47 L50 T62 P63 V65  62 P52 R53 V55 S56 Q57 D58 I61 E73 A74 A76  63 M41 I42 R44 R45 L46 D47 L50 T62 P63 I65  64 V51 G52 I54 E55 E56 D57 L60 T71 P72 M74  65 V50 T51 A53 Q54 D55 A56 Q59 H71 P72 V74  66 I41 Q42 I44 R45 E46 D47 L50 D62 K63 I65  67 M41 R42 R44 R45 F46 K47 L50 T62 P63 F65  68 I40 Q41 I43 L44 Q45 D46 L49 S61 L62 I64  69 M42 R43 I45 V46 T47 E49 V52 D64 P65 M67  70 I39 R40 E42 R43 D44 G45 L48 K60 P61 L63  71 I41 R42 I44 A45 Y46 G47 I50 E62 M63 V65  72 I58 R59 E61 R62 D63 G64 L67 K79 P80 L82  73 I43 Q44 I46 T47 T48 E50 I53 D65 A66 L68  74 I44 R45 V47 R48 E49 D50 L53 E66 D67 I69  75 I44 R45 V47 R48 E49 D50 L53 E66 D67 I69  76 L42 Q43 I45 K46 S47 E48 L51 T63 K64 T66  77 I44 H45 I47 T48 T49 E51 T54 D66 V67 L69  78 M39 K40 I42 H43 E44 D45 I48 K60 P61 T63  79 M43 L44 V46 Q47 K48 D49 I52 K64 P65 T67  80 I48 T49 G51 R52 A53 D54 L57 T68 P69 L71  81 V44 R45 V47 R48 E49 E50 L53 D66 D67 I69  82 I40 S41 F43 R44 E45 E46 L49 D61 P62 V64  83 L42 Q43 I45 K46 S47 E48 L51 T63 K64 T66  84 I56 S57 Q59 S60 K61 D62 R65 T76 P77 L79  85 L39 R40 V42 S43 H44 D46 L49 S60 D61 L63  86 M39 R40 L42 R43 K44 N45 T48 N59 E60 L62  87 I44 R45 L47 S48 D49 S50 R53 E66 E67 L69  88 M43 Q44 E46 D47 F48 D49 I52 Y64 P65 V67  89 I38 Q39 V41 K42 K43 E44 L47 D58 S59 I61  90 I46 W47 I49 K50 E51 E52 L55 K67 P68 T70  91 I48 S49 Q51 S52 K53 D54 R57 V68 P69 L71  92 I40 T41 G43 K44 A45 G46 L49 T60 P61 L63  93 I52 G53 I55 S56 Q57 D58 R61 K73 P74 A76  94 I46 R47 I49 K50 E51 D52 L55 K67 P68 T70  95 L40 Q41 R43 Q44 E45 E46 L49 G61 H62 V64  96 M42 Q43 E45 E46 V47 D48 V51 Y63 P64 V66  97 A39 R40 Y42 Y43 E44 K45 I48 K60 P61 T63  98 I98 I99 R101 E102 E103 Q104 L107 S118 D119 M121  99 L54 R55 V57 M58 H59 N60 I63 E75 P76 V78 100 I101 T102 V104 E105 R106 D107 L110 S121 D122 I124 101 L52 R53 V55 S56 R57 E58 I61 E73 I74 V76 102 I97 S98 V100 E101 R102 D103 L106 S117 D118 M120 103 L56 R57 I59 S60 Q61 N62 I65 E77 P78 V80 104 I127 T128 K130 E131 D132 D133 I136 S147 D148 V150 105 I91 T92 V94 E95 R96 E99 L102 S113 D114 V116 106 I51 R52 N54 S55 D56 G57 L60 A72 L73 A75 107 I101 T102 V104 E105 R106 D107 L110 S121 D122 I124 108 I122 V123 V125 E126 A127 D128 I131 S142 D143 V145 109 I90 T91 V93 E94 R95 E98 L101 S112 D113 V115 110 I96 R97 N99 S100 D101 G102 L105 A117 L118 A120 111 I91 T92 V94 E95 R96 E99 L102 S113 D114 V116 112 L49 R50 L52 S53 Q54 D55 I58 E70 G71 V73 113 I106 T107 V109 E110 R111 E114 L117 S128 D129 V131 114 I101 T102 V104 E105 R106 D107 L110 S121 D122 I124 115 L52 R53 V55 S56 R57 D58 I61 E73 I74 V76 116 I122 V123 V125 E126 A127 D128 I131 S142 D143 V145 117 L53 R54 V56 M57 H58 N59 I62 E74 P75 V77 129 I91 R92 N94 S95 D96 S97 L100 A112 L113 A115 SEQ ID Pos 21 Pos 22 Pos 23 Pos 24 Pos 25 Pos 26 Pos 27 Pos 28 Pos 29 Pos 30  1 S108 R116 Q119 N126 K127 Y129 L139 Q159 G210 G211  2 S108 R116 Q119 N126 K127 Y129 L139 Q159 G210 G211  3 S108 R116 Q119 N126 K127 Y129 L139 Q159 — G210  4 S108 R116 Q119 N126 K127 Y129 L139 Q159 — G210  5 S108 R116 Q119 N126 K127 Y129 L139 Q159 G210 G211  6 S78 R86 Q89 N96 K97 Y99 L109 Q129 G180 G181  7 S107 R115 L118 N125 K126 Y128 L138 Q158 G209 G210  8 S80 R88 Q91 N98 K99 Y101 L111 Q131 G182 G183  9 S79 R87 Q90 N97 K98 Y100 I110 Q130 A181 G182  10 S78 R86 Q89 K96 K97 Y99 I109 Q129 A180 G181  11 G78 086 Q89 H96 K97 Y99 V109 H129 A180 A181  12 T76 R84 Q87 N94 K95 Y97 I107 Q127 A175 G176  13 G78 H86 Q89 H96 K97 Y99 V109 Q129 A180 A181  14 T78 R86 Q89 N96 K97 Y99 L109 Q129 A177 G178  15 G78 R86 Q89 Y96 K97 Y99 I109 Q129 A180 A181  16 G78 R86 Q89 Y96 K97 Y99 I109 Q129 A180 A181  17 S78 Q86 Q89 H96 K97 Y99 V109 R129 A180 A181  18 Q78 R86 Q89 N96 K97 Y99 V109 Q129 A180 G181  19 Q73 R81 Q84 N91 K92 Y94 V104 Q124 A175 G176  20 F78 R86 Q89 N96 K97 Y99 L109 Q129 G178 G179  21 F75 R83 Q86 N93 K94 Y96 I106 Q126 G175 G176  22 S81 R89 Q92 K99 K100 Y102 L112 Q132 A182 A183  23 S83 R91 Q94 K101 K102 Y104 L114 Q134 A186 A187  24 S81 R89 Q92 K99 K100 Y102 L112 Q132 A182 A183  25 S127 R135 Q138 K145 K146 Y148 L158 Q178 A228 A229  26 S54 R62 Q65 H72 K73 Y75 I85 Q105 G151 G152  27 R108 Q116 Q119 H126 K127 Y129 I139 A159 A213 G214  28 R110 Q118 Q121 H128 K129 Y131 I141 A161 A215 G216  29 S81 R89 Q92 K99 K100 Y102 — — A152 A153  30 R110 Q118 Q121 H128 K129 Y131 I141 A161 A215 G216  31 R110 Q118 Q121 H128 K129 Y131 I141 A161 A215 G216  32 E85 R93 Q96 S103 K104 Y106 I116 R136 G188 G189  33 M131 K138 L141 A148 P149 F151 V161 R181 A226 G227  34 S80 R88 Q91 N98 K99 Y101 I111 Q131 A182 G183  35 T145 R153 Q156 K163 K164 Y166 I176 E196 A248 A249  36 R114 Q122 Q125 H132 K133 Y135 I145 K165 A219 G220  37 R75 Q83 Q86 H93 K94 Y96 I106 K126 G179 G180  38 K80 R88 Q91 S98 K99 Y101 L111 N131 G186 A187  39 F78 R86 Q89 N96 K97 Y99 I109 Q129 G178 G179  40 R75 Q83 Q86 H93 K94 Y96 I106 K126 G190 G191  41 R75 Q83 Q86 H93 K94 Y96 I106 K126 G190 G191  42 S76 R84 Q87 K94 K95 Y97 I107 Q127 A179 A180  43 R77 R85 Q88 K95 K96 Y98 L108 K128 G175 S176  44 R77 R85 Q88 K95 K96 Y98 L108 K128 G175 S176  45 S108 R116 Q119 N126 K127 Y129 L139 Q159 G210 G211  46 S108 R116 Q119 N126 K127 Y129 L139 Q159 — G210  47 — — — — — — — A13 A67 G68  48 R10 E18 L21 H28 K29 Y31 — K42 A96 G97  49 F69 R77 Q80 Q87 K88 Y90 L100 Q120 G168 G169  50 — — — — — — — — — —  51 E66 E74 C77 A85 N86 F88 L98 R118 A162 G163  52 T66 H74 R77 D85 T86 Y88 L98 R118 A161 G162  53 T66 H74 R77 D85 T86 Y88 L98 R118 A161 G162  54 — — — — — — — — — —  55 E66 E74 C77 A85 N86 F88 L98 R118 A162 G163  56 Q65 Q73 K76 N84 K85 Y87 L97 Q117 A160 G161  57 K82 N90 R93 K101 N102 Y104 A114 K134 A178 G179  58 A70 D78 K81 K89 N90 Y92 M102 N122 A166 G167  59 Q67 K75 L78 N86 K87 Y89 L99 R119 A163 G164  60 D67 K75 I78 K86 K87 F89 V99 R119 G163 A164  61 R67 E75 R78 A85 T86 F89 L99 R119 A163 G164  62 S78 R86 Q89 H96 K97 Y99 I109 Q129 G175 G176  63 K67 S75 M78 A85 S86 Y89 L99 R119 A163 G164  64 A76 V84 I87 A94 K95 F98 L108 H128 A170 G171  65 D76 K85 M88 A95 K96 Y99 L109 H129 S176 G177  66 A67 D75 I78 S85 K86 F89 L99 R119 A163 G164  67 E67 A75 R78 A85 R86 Y89 L99 R119 A161 G162  68 D66 E75 I78 A85 Q86 Y89 V99 R119 A163 G164  69 A69 R77 L80 G87 K88 Y91 I101 R121 S164 G165  70 A65 E73 R76 A83 K84 F87 A97 R117 A161 G162  71 R67 R75 Q78 N85 K86 F88 I98 R118 A173 A174  72 A84 E92 R95 A102 K103 F106 A116 R136 A180 G181  73 Q70 R78 I81 S88 K89 F92 I102 R122 A166 G167  74 T71 R79 I82 S89 K90 Y93 V103 D123 A167 S168  75 T71 R79 I82 S89 K90 Y93 V103 D123 A167 S168  76 D68 L76 A79 S86 S87 F90 L100 R120 A165 A166  77 Q71 R79 I82 G89 R90 Y93 I103 R123 A167 G168  78 N65 E73 L76 A83 R84 F87 L97 R117 A161 G162  79 D69 E77 L80 A87 R88 F91 L101 R121 A165 G166  80 G73 K81 L84 L91 P92 Y94 V104 R124 A176 G177  81 A71 R79 I82 S89 K90 Y93 V103 D123 A167 S168  82 T66 R74 L77 A84 S85 Y88 V98 G118 A164 G165  83 D68 L76 A79 S86 S87 F90 L100 R120 A165 A166  84 N81 T89 L92 L99 P100 Y102 V112 R132 A177 G178  85 E65 V73 I76 S83 K84 Y87 L97 N117 A161 G162  86 Q64 E72 L75 N82 N83 Y86 V96 R116 G159 G160  87 K71 A79 I82 G89 K90 F93 L103 R123 A172 S173  88 E69 K77 L80 A87 K88 L91 L101 R121 A164 G165  89 D63 E71 M74 S81 K82 Y85 L95 S115 A158 G159  90 D72 S80 L83 A90 R91 F94 L104 R124 A168 G169  91 N73 A81 L84 L91 P92 Y94 V104 R124 A169 G170  92 K65 R73 L76 L83 P84 F86 V96 R116 A168 G169  93 E78 E86 L89 Y96 R97 L100 V110 R138 T187 S188  94 D72 D80 L83 A90 R91 F94 L104 R124 A170 G171  95 E66 E74 I77 A84 K85 Y88 V97 R117 A162 G163  96 E68 D76 L79 A86 K87 L90 L100 R120 A163 G164  97 E65 E73 L76 A83 R84 F87 L97 R117 A160 G161  98 T123 K131 L134 A141 P142 F144 V154 R174 A219 G220  99 S80 R88 Q91 K110 K111 Y113 I123 Q143 A195 A196 100 T126 K134 L137 A144 P145 F147 V157 R177 A222 G223 101 S78 Q86 E89 N96 K97 Y99 I109 Q129 A180 G181 102 T122 K130 L133 A140 P141 F143 V153 R173 A218 G219 103 R82 R90 Q93 H100 K101 Y103 V113 0133 G179 G180 104 T152 K160 L163 A170 P171 F173 V183 R203 A248 G249 105 T118 K126 L129 A136 P137 F139 V149 R169 A214 G215 106 R77 E85 L88 H95 K96 Y98 I108 K128 A182 G183 107 T126 K134 L137 A144 P145 F147 V157 R177 A222 G223 108 T147 K155 L158 A165 P166 F168 V178 R198 A243 G244 109 T117 K125 L128 A135 P136 F138 V148 R168 A213 G214 110 R122 Q130 L133 H140 K141 Y143 I153 K173 A227 G228 111 T118 K126 L129 A136 P137 F139 V149 R169 A214 G215 112 F75 R83 Q86 N93 K94 Y96 I106 Q126 G175 G176 113 T133 K141 L144 A151 P152 F154 V164 R184 A229 G230 114 T126 K134 X137 A144 P145 F147 V157 R177 A222 G223 115 S78 Q86 Q89 N96 K97 Y99 I109 Q129 A180 G181 116 T147 K155 L158 A165 P166 F168 V178 R198 A243 G244 117 S79 R87 Q90 K97 K98 Y100 I110 Q130 A182 A183 129 R117 E125 L128 H135 K136 Y138 I148 K168 A222 G223 SEQ ID Pos 31 Pos 32 Pos 33 Pos 34 Pos 35 Pos 36 Pos 37 Pos 38 Pos 39 Pos 40  1 E224 L245 S246 K248 E249 G252 E253 N254 A255 I257  2 E224 L245 S246 K248 E249 G252 E253 N254 A255 I257  3 E223 L244 S245 K247 E248 G251 E252 N253 A254 I256  4 E223 L244 S245 K247 E248 G251 E252 N253 A254 I256  5 D224 L245 S246 K248 E249 G252 G253 E254 A256 I258  6 E194 L215 S216 K218 E219 G222 G223 G224 A226 I228  7 E223 S244 S245 K247 E248 G251 E252 K253 S255 N257  8 E196 S217 A218 R220 E221 G224 E225 T226 S229 E231  9 E195 S216 T217 R219 G220 G223 E224 T225 S228 V230  10 E194 S215 A216 R218 E219 G222 E223 R224 T226 E228  11 E194 F215 A216 R218 E219 G222 E223 N224 A227 R229  12 D189 S210 A211 K213 G214 G217 E218 T219 S222 E224  13 E194 F215 G216 K218 D219 G222 E223 T224 V227 R229  14 E191 S212 S213 K215 E216 G219 D220 T221 S224 E226  15 E194 S215 A216 K218 E219 A222 E223 A224 S227 E229  16 E194 S215 A216 K218 E219 A222 E223 A224 S227 E229  17 E194 F215 G216 R218 D219 G222 E223 T224 A227 R229  18 E194 S215 T216 K218 E219 G222 V223 R224 T227 G229  19 E189 S210 T211 K213 E214 G217 V218 R219 T222 G224  20 E192 S213 P214 N216 E217 G220 G221 P222 T225 A227  21 D189 S210 P211 K213 E214 G217 G218 P219 T222 I224  22 D196 A217 A218 G220 G221 R224 D225 T226 S229 G231  23 D200 A221 A222 G224 G225 G228 E229 A230 S233 G235  24 D196 A214 A215 G217 G218 R221 D222 T223 S226 G228  25 D242 A260 A261 G263 G264 R267 D268 T269 S272 G274  26 E165 S186 S187 G189 G190 K193 — — P194 G196  27 A227 T248 A249 G251 D252 K255 T256 G257 L260 G262  28 A229 T250 A251 G253 D254 K257 T258 R259 S262 A264  29 D166 A187 A188 G190 G191 R194 D195 T196 S199 G201  30 A229 A250 A251 G253 D254 K257 T258 R259 S262 G264  31 A229 A250 A251 G253 D254 K257 T258 R259 S262 G264  32 E202 L223 D224 R226 K227 T231 E232 R233 A234 V236  33 K240 E261 R262 K264 N265 P269 R270 D271 P272 L274  34 D196 S217 A218 K220 E221 K226 K227 G228 S229 E231  35 D283 A304 A305 — — — — — — —  36 G233 T254 A255 G257 D258 A263 D264 T265 S266 G268  37 A193 S214 T215 G217 D218 G223 G224 A225 S226 G228  38 E200 S221 R222 K224 A225 A229 K230 H231 V232 H234  39 E192 S213 P214 K216 E217 P222 P223 K224 T225 V227  40 A204 S225 T226 G228 D229 G234 G235 A236 S237 G239  41 A204 S225 T226 G228 D229 G234 G235 A236 S237 G239  42 D193 A214 A215 G217 G218 T223 R224 T225 S226 G228  43 E189 S210 K211 K213 K214 S227 D228 F229 P230 R232  44 E189 S210 K211 K213 K214 S227 D228 F229 P230 R232  45 E224 — — — — — — — — —  46 E223 — — — — — — — — —  47 A81 A102 A103 G105 D106 R111 H112 D113 S114 G116  48 G110 T131 A132 G134 D135 G140 G141 A142 S143 G145  49 D182 S203 A204 R206 G207 N212 K213 G214 S215 T217  50 — S2 S3 G5 E6 H11 E12 A13 T14 R16  51 K176 K197 Q198 Y200 H201 — — — — —  52 R175 T196 S197 D199 G200 — — — — —  53 R175 T196 S197 D199 G200 — — — — —  54 — — — — — — — — —  55 K176 K197 0198 Y200 H201 — — — — —  56 K174 E195 R196 K198 R199 — — — — —  57 K192 A213 R214 A216 R217 — — — — —  58 K180 E201 R202 K204 R205 — — — — —  59 K177 E198 R199 K201 R202 — — — — —  60 P177 E198 K199 A201 A202 — — — — —  61 R177 T198 G199 T201 D202 S205 T206 A207 T208 R210  62 — — — — — — — — — —  63 K177 E198 R199 Q201 R202 E205 S206 K207 Q208 A210  64 K184 A205 A206 A208 N209 P212 V213 T214 L215 A217  65 K190 K211 T212 P214 D215 Y218 K219 P220 Y221 G223  66 K177 D198 Q199 N201 S202 S205 K206 H207 K208 Q210  67 R175 E196 R197 R199 R200 A203 P204 K205 I206 A208  68 K177 A198 A199 A201 N202 P205 K206 A207 K208 Y210  69 S178 A199 K200 A202 A203 K206 K207 A208 K209 I211  70 L175 A196 R197 A199 T200 A203 G204 P205 A206 K208  71 — — — — — — — — — —  72 L194 A215 R216 A218 T219 A222 G223 P224 A225 K227  73 S180 P201 K202 Q204 V205 P208 K209 M210 R211 R213  74 L181 K202 R203 K205 R206 G209 S210 S211 P212 G214  75 L181 K202 R203 K205 R206 G209 S210 S211 P212 G214  76 S179 Q200 P201 E203 K204 K207 S208 — — —  77 S181 Q202 K203 L205 I206 P209 K210 M211 R212 R214  78 A175 E196 A197 K199 S200 K203 S204 G205 P206 G208  79 A179 E200 K201 D203 A204 G207 P208 G209 — —  80 R190 N211 K212 A214 P215 T218 P219 D220 P221 1223  81 L181 K202 R203 K205 R206 G209 S210 A211 P212 G214  82 L178 K199 Q200 — — G204 T205 A206 Y207 K209  83 S179 Q200 P201 E203 K204 K207 S208 — — —  84 R191 Q212 R213 Q215 P216 A219 A220 I221 Q222 P224  85 K175 G196 A197 R199 R200 — — — — —  86 E173 R194 G195 — — — — — — —  87 R186 Q207 K208 — — — — — — A210  88 K178 A199 P200 — — — — K201 S202 R204  89 A172 N193 T194 — — — — — — —  90 R182 K203 K204 E207 A208 G211 K212 A213 V214 S216  91 R183 S204 R205 H216 Q217 A220 N221 S222 P223 Q225  92 R182 Q203 R204 Q206 Q207 P210 T211 D212 P213 L215  93 R201 Q222 T223 — — — K224 S225 A226 A228  94 R184 K205 K206 E209 I210 G213 K214 Q215 V216 S218  95 R176 K197 K198 — — — — S199 P200 T202  96 K177 Q198 P199 — — — — K200 T201 R203  97 T174 E195 A196 — K197 G200 A201 K202 S203 G205  98 K233 A254 K255 A258 P259 T262 R263 D264 P265 L267  99 D209 A230 A231 K235 N236 T239 K240 S241 S242 G244 100 R236 E257 R258 T261 P262 P265 R266 D267 P268 L270 101 E194 S215 A216 T219 N220 E223 T224 K225 N226 V228 102 K232 A253 K254 S257 T258 P261 R262 D263 P264 L266 103 E193 S214 S215 — R216 K219 K220 S221 P222 G224 104 R262 E283 K284 K287 P288 P291 R292 D293 P294 L296 105 K228 D249 R250 N253 P254 P257 R258 D259 P260 L262 106 A196 T217 A218 S222 T223 G226 S227 A228 V229 G231 107 R236 E257 R258 T261 P262 P265 R266 D267 P268 L270 108 K257 E278 R279 N282 P283 P286 R287 D288 Q289 L291 109 K227 D248 K249 N252 P253 P256 R257 D258 P259 L261 110 G241 T262 A263 S267 A268 G271 G272 A273 S274 G276 111 K228 D249 K250 N253 P254 P257 R258 D259 P260 L262 112 D189 S210 P211 K215 K216 P219 P220 R221 T222 1224 113 K243 D264 K265 N268 P269 P272 R273 D274 P275 L277 114 R236 E257 R258 T261 P262 P265 R266 D267 P268 L270 115 G194 S215 A216 T219 N220 E223 T224 K225 N226 V228 116 K257 E278 R279 N282 P283 P286 R287 D288 Q289 L291 117 D196 A217 A218 K222 N223 T226 K227 S228 S229 G231 129 G236 T257 A258 G260 D261 G264 S265 A266 V267 G269 SEQ ID Pos 41 Pos 42 Pos 43 Pos 44 Pos 45 Pos 46 Pos 47 Pos 48 Pos 49 Pos 50  1 K259 P260 V262 G264 D286 Q291 P305 G308 N309 Q323  2 K259 P260 V262 G264 D286 Q291 P305 G308 N309 Q323  3 K258 P259 V261 G263 D285 Q290 P304 G307 N308 Q322  4 K258 P259 V261 G263 D285 Q290 P304 G307 N308 Q322  5 K260 P261 V263 G265 D287 Q292 P306 G309 N310 Q324  6 K230 P231 V233 G235 D257 Q262 P276 G279 N280 Q294  7 K259 P260 V262 G264 D286 Q291 L305 E308 N309 Q323  8 K233 K234 Q236 G238 D260 K265 S279 E282 N283 K297  9 K232 K233 Q235 G237 D259 E264 S278 E281 N282 Q296  10 R230 K231 L233 G235 D257 K262 S276 E279 N280 Q294  11 N231 K232 K234 G236 D258 N263 S277 Q280 N281 —  12 G226 K227 Q229 G231 D253 N258 S272 E275 N276 Q290  13 N231 K232 Q234 G236 D258 N263 S277 Q280 N281 —  14 G228 K229 Q231 G233 H255 N260 S274 E277 N278 Q292  15 K231 H232 Q234 G236 D258 K263 S277 E280 N281 Q294  16 K231 H232 Q234 G236 D258 K263 S277 E280 N281 Q294  17 S231 K232 Q234 G236 D258 N263 S277 E280 N281 —  18 S231 K232 Q234 G236 G258 R263 Y276 Q279 N280 L294  19 S226 K227 R229 G231 G253 R258 Y271 Q274 N275 L289  20 K229 K230 Q232 G234 D256 N261 S275 D278 S279 E293  21 K226 K227 Q229 G231 D253 N258 S272 D275 S276 E290  22 K233 K234 S236 G238 D260 D265 Q279 E280 N281 Q295  23 K237 R238 S240 R242 D264 D269 Q283 E284 N285 Q299  24 K230 K231 S233 G235 D257 D262 Q276 E277 N278 Q292  25 K276 K277 S279 G281 D303 D308 Q322 E323 N324 Q338  26 S198 K199 R201 G203 H225 Q230 A244 G246 N247 Q261  27 R264 R265 R267 A269 D291 G296 P310 G313 G314 D328  28 R266 R267 R269 V271 D293 G298 P312 G315 G316 D330  29 K203 K204 S206 G208 D230 D235 Q249 E250 N251 Q265  30 R266 R267 R269 V271 D293 G298 P312 G315 R316 D330  31 R266 R267 R269 V271 D293 G298 P312 G315 R316 D330  32 K238 R239 P241 G243 E265 N270 P284 D287 S288 S307  33 T276 P277 G279 T281 D302 S307 — G319 G320 V334  34 K233 K234 Q236 G238 D260 E265 S279 E282 N283 S299  35 — — — — D322 D327 Q341 E342 N343 L359  36 G270 R271 K273 A275 G297 G302 S316 D319 G320 Q339  37 G230 R231 K233 V235 G257 G262 S276 G279 G280 Q299  38 Q236 K237 Q239 G241 E263 H268 P282 N285 N286 Q305  39 K229 K230 Q232 G234 D256 N261 S275 D278 S279 E295  40 G241 R242 K244 V246 G268 G273 S287 G290 G291 Q310  41 G241 R242 K244 V246 G268 G273 S287 G290 G291 Q310  42 K230 K231 S233 G235 D257 D262 Q276 E277 N278 L294  43 R234 P235 R237 G239 E261 N266 P280 Q283 S284 K302  44 R234 P235 R237 G239 E261 N266 P280 Q283 S284 K302  45 — — — — — — — — — —  46 — — — — — — — — — —  47 R118 R119 R121 V123 D145 G150 P164 G167 R168 Q187  48 G147 R148 K150 A152 S174 G179 S193 D196 G197 A216  49 E219 R220 K222 K224 N246 R251 S265 D268 S269 T286  50 P18 K19 Q21 G23 E45 H50 D64 S67 N68 Q87  51 — — R205 S207 H229 N234 — N246 P247 R261  52 P203 D204 P206 G208 D229 N234 — T245 A246 R261  53 P203 D204 P206 G208 D229 N234 — T245 A246 R261  54 — — — — — — — — — —  55 — — R205 S207 H229 N234 — N246 P247 R261  56 A203 K204 R206 K208 E229 N234 P242 K245 G246 Q260  57 — — R221 G223 G245 G250 V257 D260 K261 Q275  58 S209 K210 K212 K214 D235 S240 — E251 K252 E262  59 S206 K207 S209 R211 D232 S237 — E248 G249 1263  60 — F205 K207 V209 D230 G235 — D246 G247 E261  61 — — — R211 A233 E237 — G249 T250 —  62 — — — — — — — — — —  63 — — — K211 N233 S237 — K249 Q250 I264  64 — — K218 K220 S242 D250 — G262 G263 T276  65 — K224 L226 A228 P252 H260 — G272 A273 G280  66 — — K211 R213 G235 E240 — K253 R254 G269  67 — — — R209 T231 A235 — A246 A247 L261  68 — — — — P232 G236 — D247 M248 K258  69 P213 K214 R216 V218 Q240 N245 — T256 G257 I271  70 R210 P211 R213 R215 G237 G242 A253 G256 A257 G272  71 — — — — — — — — — —  72 R229 P230 R232 R234 G256 G261 A272 G275 A276 G291  73 — — — — G235 E240 — S251 W252 I267  74 — — — — D236 N240 — E251 G252 M266  75 — — — — D236 N240 — E251 G252 M266  76 — — — R209 P231 G235 — V247 Q248 E263  77 — — — — G236 G241 — G251 T252 I267  78 — — G210 V212 S234 E238 — K249 G250 E264  79 — — — V211 E233 G237 — G248 K249 E263  80 K225 T226 P228 E230 R252 N256 — Q268 T269 E284  81 — — — — D236 N240 — E251 G252 M266  82 — — — R210 R232 G236 — D247 H248 G262  83 — — — R209 P231 G235 — V247 Q248 E263  84 — P225 R227 Q229 S251 G255 — E266 L267 A282  85 — — — R201 — R226 — G238 G239 F252  86 — — F197 R199 I219 D224 — — K236 E250  87 A212 P213 A216 E218 D239 N244 — — F257 I270  88 K206 R207 T209 E211 D233 G238 — — K250 I260  89 — — R196 L198 — N222 — — — T244  90 A218 G219 G221 V223 A245 G250 — — P263 I273  91 P227 P228 R230 Q232 S253 G258 R272 — G274 V293  92 T217 V218 R220 E222 E243 N248 — — T261 V275  93 G230 A231 Y233 L235 T257 E263 — — — Q289  94 A220 G221 G223 V225 D247 G252 — — — L275  95 K204 T205 — — D227 N232 — — W245 W254  96 K205 R206 T208 A210 E232 N237 — — W250 I259  97 A207 G208 G210 V212 E233 E238 — — K250 E264  98 K269 P270 G272 T274 D295 S300 — — E313 V327  99 K246 K247 S249 G251 E273 D278 E292 — N294 L310 100 K272 P273 G275 T277 S298 S303 — — G316 L330 101 R230 K231 L233 G235 D257 K262 S276 E279 N280 S296 102 T268 P269 G271 T273 S294 S299 — — G312 L326 103 S226 K227 R229 G231 H253 Q258 T272 G274 N275 Q291 104 K298 P299 G301 T303 S324 S329 — — E342 V356 105 T264 P265 G267 T269 S290 S295 — — G308 V322 106 G233 R234 K236 V238 G260 A265 S279 N282 G283 Q302 107 K272 P273 G275 T277 S298 S303 — — G316 L330 108 K293 P294 G296 T298 S319 S324 — — E337 V351 109 A263 P264 G266 T268 S289 S294 — — G307 V321 110 G278 R279 K281 A283 T305 G310 S324 D327 G328 Q347 111 A264 P265 G267 T269 S290 S295 — — G308 V322 112 K226 K227 Q229 G231 D253 N258 S272 D275 S276 E292 113 A279 P280 G282 T284 S305 S310 — — G323 V337 114 K272 P273 G275 T277 S298 S303 — — G316 L330 115 R230 K231 L233 G235 D257 K262 S276 E279 N280 S296 116 K293 P294 G296 T298 S319 S324 — — E337 V351 117 K233 K234 S236 G238 D260 D265 Q279 — N281 L297 129 G271 R272 K274 A276 G298 G303 S317 D320 G321 H340 SEQ ID Pos 51 Pos 52 Pos 53 Pos 54 Pos 55 Pos 56 Pos 57 Pos 58 Pos 59 Pos 60 Pos 61 Pos 62 Pos 63  1 R335 M343 F345 T358 D372 K373 S387 H391 N392 L400 S412 M414 C415  2 R335 M343 F345 T358 D372 K373 S387 H391 N392 L400 S412 M414 C415  3 R334 M342 F344 T357 D371 K372 S386 H390 N391 L399 S411 M413 C414  4 R334 M342 F344 T357 D371 K372 S386 H390 N391 L399 S411 M413 C414  5 R336 M344 F346 T359 D373 K374 S388 H392 N393 L401 S413 M415 C416  6 R306 M314 F316 S329 D343 K344 S358 H362 N363 L371 S383 M385 C386  7 N335 M343 V345 S358 T372 N373 S387 H391 N392 L400 S412 V414 Y415  8 C311 M319 K321 S334 E348 N349 S363 Q367 N368 L376 N388 L390 Y391  9 C310 T318 N320 S333 D347 N348 S362 Q366 N367 L375 N387 L389 Y390  10 C308 M316 G318 S331 E345 N346 S360 Q364 N365 L373 N385 L387 Y388  11 Y305 T313 R315 S328 E342 N343 S357 K361 N362 L370 S382 L384 Y385  12 C304 T312 R314 V327 E341 N342 S356 K360 N361 L369 S381 L383 Y384  13 G305 K313 K315 T328 E342 N343 S357 Q361 N362 L370 S382 M384 H385  14 C306 T314 R316 N329 E343 N344 S358 E362 N363 L371 S383 Q385 Y386  15 C308 T316 G318 1331 E345 N346 S360 Q364 N365 L373 K385 L387 F388  16 C308 T316 G318 1331 E345 N346 S360 Q364 N365 L373 K385 L387 F388  17 G305 T313 R315 T328 E342 N343 S357 Q361 N362 L370 S382 L384 H385  18 C306 M314 G316 A329 E343 S344 S358 Q362 N363 L371 N383 E385 Y386  19 C301 M309 G311 A324 E338 S339 S353 Q357 N358 L366 N378 E380 Y381  20 C307 A315 R317 D330 E344 N345 S359 Q363 H364 L372 N384 V386 Y387  21 C304 A312 R314 D327 E341 S342 S356 K360 H361 L369 N381 V383 Y384  22 C309 M317 G319 N332 E346 K347 S361 K365 H366 L374 S386 V388 H389  23 C313 T321 G323 N336 E350 K351 S365 K369 H370 L378 S390 L392 H393  24 C302 M310 G312 N325 E339 K340 S354 K358 H359 L367 S367 V381 H382  25 C348 M356 G358 N371 E385 K386 S400 K404 H405 L413 S425 V427 H428  26 N274 T282 T284 S297 E311 N312 A326 E330 N331 L339 E351 V353 Y354  27 S345 R353 G355 N368 E382 D383 Y397 Q401 H403 L411 D423 Q425 Y426  28 S347 T355 G357 D370 E384 D385 Y399 Q403 H405 L413 D425 Q427 Y428  29 C279 M287 G289 N302 E316 K317 S331 K335 H336 L344 S356 V358 H359  30 S347 T355 G357 D370 D384 D385 Y399 Q403 H405 L413 D425 Q427 Y428  31 S347 T355 G357 D370 D384 D385 Y399 Q403 H405 L413 D425 Q427 Y428  32 G319 T327 K329 T342 E356 N357 S371 E375 G376 L384 S396 Q398 Y399  33 Y346 P354 S356 Y367 D381 S382 R402 — — I412 P424 R426 V427  34 C311 N319 G321 S334 E348 D349 S363 Q367 N368 L376 K388 S390 Y391  35 C371 M379 G381 K394 E408 K409 S423 K427 H428 L436 S448 L450 H451  36 S351 T359 G361 D374 E388 D389 Y403 K408 Y409 L417 N429 Q431 H432  37 S311 T319 G321 D334 E348 D349 Y363 K368 H369 L377 N389 Q391 Y392  38 — — — T315 Q329 D330 S344 K348 N349 L357 T369 Q371 Y372  39 C307 A315 R317 D330 E344 S345 S359 K363 H364 L372 N384 V386 Y387  40 S322 T330 G332 D345 E359 D360 Y374 K379 H380 L388 N400 Q402 Y403  41 S322 T330 G332 D345 E359 D360 Y374 K379 H380 L388 N400 Q402 Y403  42 C306 V314 G316 K329 E343 K344 S358 K362 H363 L371 S383 L385 H386  43 D314 V322 D324 I337 D351 S352 S366 A371 N372 L380 E392 Q394 L395  44 D314 V322 D324 I337 D351 S352 S366 A371 N372 L380 E392 Q394 L395  45 — — — — — — — — — — — — —  46 — — — — — — — — — — — — —  47 S199 T207 G209 D222 D236 D237 Y251 K256 H257 L265 D277 Q279 Y280  48 S228 T236 G238 D251 E265 D266 F280 K285 H286 L294 N306 Q308 Y309  49 S298 — — — — — — — — — — — —  50 R99 S107 0109 M122 E136 N137 S151 G155 S156 L164 K176 Q178 Y179  51 H273 — — E293 E307 Q308 A322 — — L333 E345 H347 V348  52 H273 — — T293 D307 A308 P322 — — I333 E345 H347 V348  53 H273 — — T293 D307 A308 P322 — — I333 E345 H347 V348  54 S6 T14 G16 D29 D43 D44 Y58 K63 H64 L72 D84 Q86 Y87  55 H273 — — E293 E307 Q308 A322 — — L333 E345 H347 V348  56 Y272 S280 L282 L293 D307 D308 A322 — — I332 Q344 K346 V347  57 P287 G295 L297 E309 E323 Q324 A338 — — L348 L360 H362 V363  58 H274 — — I296 D310 Q311 K325 — — I335 E347 Y349 V350  59 Y275 S282 F284 Y296 K310 N311 E325 — — L335 N347 F349 A350  60 P273 — — Q295 Q309 D310 K324 — — L334 D346 Y348 I349  61 H272 — — P292 A306 D307 S321 Q325 — — A344 H346 V347  62 — — — — — — — — — — — — —  63 Y276 F282 E284 Y297 K311 D312 S326 K330 — I336 E348 M350 A351  64 H286 L292 E294 E307 S321 E322 S336 K340 — L346 A358 K360 V361  65 H292 M301 A303 K316 S330 Q331 T345 R349 — N355 P368 K370 V371  66 H281 L288 E290 D303 E317 Q318 F332 S336 — L342 E354 H356 V357  67 Y273 L279 R281 V294 T308 A309 E323 R327 — I333 Q345 F347 V348  68 H270 — — D287 S301 A302 E316 R320 — L326 E338 H340 G341  69 Y283 I289 E291 R304 N318 Q319 S333 R337 — L343 A355 R357 V358  70 E294 I301 A303 E316 E330 Q331 S345 R349 — L355 A367 H369 I370  71 — — — T211 R225 M226 — — — M233 — — —  72 E310 I317 A319 E332 E346 Q347 S361 R365 — L371 A383 H385 I386  73 F278 I284 S286 P299 D313 Q314 S328 R332 — L338 H350 H352 V353  74 P278 L284 G286 P299 E313 Q314 T328 R332 — I338 E350 K352 V353  75 P278 L284 G286 P299 E313 Q314 T328 R332 — I338 E350 K352 V353  76 H375 V283 I285 Y298 R312 Q313 E327 R331 — L337 E349 S351 V352  77 Y279 V285 A287 P300 D314 Q315 S329 R333 — L339 P351 R353 V354  78 Y276 L282 D284 E297 K311 G312 R326 R330 — L336 S348 K350 A351  79 Y275 V281 N283 E296 K310 G311 R325 R329 — L335 E347 K349 A350  80 G295 L301 P303 P316 S330 A331 R345 I349 — I355 E367 W369 Q370  81 P278 L284 G286 P299 E313 Q314 T328 R332 — I338 D350 K352 V353  82 H274 W281 E283 E296 E310 Q311 S325 P329 — L335 D347 H349 Q350  83 H275 V283 I285 Y298 R312 Q313 E327 R331 — L337 E349 S351 V352  84 Y293 L299 E301 L314 E328 A329 R343 L347 — I353 Q365 Y367 H368  85 F264 L270 P272 H285 E299 D300 K314 P318 — I324 D336 Q338 V339  86 H262 I268 F270 N283 A297 E298 W312 — — I322 S334 E336 V337  87 A282 I288 P290 P303 D317 R318 S332 — — L342 D354 H356 V357  88 Y272 L278 F280 Y293 T307 G308 S321 — — L331 E343 G345 A346  89 Y256 I262 D264 N277 K291 D292 K306 — — I316 K328 Q330 F331  90 H285 V291 G293 P306 E320 R321 K335 — — L345 E357 K359 V360  91 Y305 L311 D313 P326 E340 A341 R355 — — I365 Q377 Y379 H380  92 Y287 L293 N295 P308 D322 A323 R337 — — I347 Q359 W361 Q362  93 P301 L307 A309 E322 E336 N337 A351 — — A361 E373 Q375 V376  94 Y287 L293 G295 P308 E322 K323 K337 — — L347 E359 K361 V362  95 G266 L272 P274 P287 M301 Q302 R316 — — L326 A338 Q340 V341  96 Y271 L277 F279 Y292 T306 G307 S320 — — L330 K342 G344 A345  97 Y276 L282 E284 E297 R311 G312 K326 — — L336 E348 K350 A351  98 H339 L345 P347 Y360 E374 A375 R395 — — I405 P417 R419 V420  99 C322 M330 G332 K345 E359 K360 S374 K378 H379 L387 S399 L401 H402 100 H342 L348 P350 Y363 E377 A378 R398 — — I408 S420 R422 V423 101 C308 T316 G318 S331 E345 N346 S360 Q364 N365 L373 N385 L387 Y388 102 Y338 L344 P346 Y359 E373 A374 R394 — — I404 P416 R418 V419 103 G303 T311 T313 S326 E340 N341 A355 E359 N360 L368 E380 L382 Y383 104 Y368 L374 P376 H389 E403 A404 R424 — — I434 P446 R448 T449 105 Y334 L340 P342 Y355 E369 A370 R390 — — I400 A412 R414 V415 106 S314 T322 G324 D337 E351 D352 Y366 K371 H372 L380 N392 Q394 H395 107 H342 L348 P350 Y363 E377 A378 R398 — — I408 S420 R422 V423 108 Y363 L369 P371 Y384 E398 A399 R419 — — I429 P441 R443 I444 109 Y333 L339 P341 Y354 E368 A369 R389 — — I399 A411 R413 V414 110 S359 T367 G369 D382 E396 D397 F411 K416 H417 L425 N437 Q439 H440 111 Y334 L340 P342 Y355 E369 A370 R390 — — I400 A412 R414 V415 112 C304 A312 R314 D327 E341 S342 S356 K360 H361 L369 N381 V383 Y384 113 Y349 L355 P357 Y370 E384 A385 R405 — — I415 A427 R429 V430 114 H342 L348 P350 Y363 E377 A378 R398 — — I408 S420 R422 V423 115 C308 T316 G318 S331 E345 N346 S360 Q364 N365 L373 N385 L387 Y388 116 Y363 L369 P371 Y384 E398 A399 R419 — — I429 P441 R443 I444 117 C309 M317 G319 K332 E346 K347 T361 K365 H366 L374 S386 L388 H389 129 S352 T360 G362 D375 E389 D390 Y404 Q408 H410 L418 N430 Q432 H433 SEQ ID Pos 64 Pos 65 Pos 66 Pos 67 Pos 68 Pos 69 Pos 70 Pos 71 Pos 72 Pos 73 Pos 74 Pos 75 Pos 76  1 R425 K428 N431 S433 T434 D435 E436 Q447 T451 D453 S464 A466 D482  2 R425 K428 N431 S433 T434 D435 E436 Q447 T451 D453 S464 A466 D482  3 R424 K427 N430 S432 T433 D434 E435 Q446 T450 D452 S463 A465 D481  4 R424 K427 N430 S432 T433 D434 E435 Q446 T450 D452 S463 A465 D481  5 R426 K429 K432 S434 T425 D436 E437 Q448 R452 D454 D465 A467 D483  6 R396 K399 N402 S404 T405 D406 E407 Q418 T422 D424 S435 A437 D453  7 R425 E428 K431 S433 T434 D435 E426 Q447 T451 G453 S363 A466 E482  8 R401 E404 K407 S409 T410 D411 E412 Q423 A427 G429 S440 A442 D458  9 R400 E403 K406 S408 T409 D410 D411 Q422 A426 G428 S439 A441 D457  10 R398 E401 K404 S406 T407 D408 E409 Q420 V424 G426 S437 A439 E455  11 Q395 E398 Q401 S403 T404 D405 E406 K417 A421 G423 S434 G436 D452  12 R394 E397 K400 S402 T403 D404 E405 H416 A420 G422 S433 A435 E451  13 R395 E398 Q401 S403 T404 D405 E406 K417 A421 G423 S434 G436 D452  14 R396 E399 K402 S404 K405 D406 E407 Q418 A422 G424 S435 A437 E453  15 R398 E401 K404 S406 T407 D408 E409 Q420 V424 G426 S437 A439 E455  16 R398 E401 K404 S406 T407 D408 E409 Q420 V424 G426 S437 A439 E455  17 R395 E398 Q401 S403 T404 D405 E406 K417 A421 G423 S434 G436 D452  18 R396 E399 K402 S404 T405 D406 E407 Q418 V422 G424 S435 A437 E453  19 R391 E394 K397 S399 T400 D401 E402 Q413 V417 G419 S430 A432 E448  20 R397 E400 K403 S405 R406 T407 E408 Q419 A423 G425 S436 A438 D454  21 R394 E397 K400 S402 R403 T404 E405 Q416 A420 G422 S433 A435 D451  22 R399 E402 K405 S407 T408 D409 E410 R421 V425 G427 R438 A440 D456  23 R403 E406 K409 S411 T412 D413 E414 R425 V429 G431 R442 A444 D460  24 R392 E395 K398 S400 T401 D402 E403 R414 V418 G420 R431 A433 D449  25 R438 E441 K444 S446 T447 D448 E449 R460 V464 G466 R477 A479 D495  26 R364 E367 K370 S372 R373 D374 E375 Q386 T390 G392 S403 A405 E421  27 H436 D439 G442 P444 T445 S446 I447 K458 V462 G464 R475 A477 E493  28 H438 D441 G444 P446 T447 S448 I449 K460 V464 G466 G477 A479 E495  29 R369 E372 K375 S377 T378 D379 E380 R391 V395 G397 R408 A410 D426  30 H438 D441 G444 P446 T447 S448 I449 K460 V464 G466 G477 A479 E495  31 H438 D441 G444 P446 T447 S448 I449 K460 V464 G466 G477 A479 E495  32 R409 D412 G415 S417 L418 E419 E420 K431 V435 G437 S448 A450 D466  33 T437 A440 S443 T445 E446 S447 E448 K459 K463 A467 P478 A480 T496  34 — — — — — — — — C404 — S410 A412 E428  35 R461 E464 K467 S469 T470 D471 E472 R483 V487 G489 R500 A502 D518  36 H442 D445 A448 P450 T451 A452 I453 K464 V468 G470 K481 A483 G499  37 H402 D405 G408 P410 T411 A412 I413 K424 V428 G430 R441 A443 A459  38 R382 K385 K388 Q390 L391 K392 D393 K404 V408 S410 N421 A423 D439  39 R397 E400 K403 — — — — — — — — — —  40 H413 D416 G419 P421 T422 A423 I424 K435 V439 G441 R452 A454 A470  41 H413 D416 G419 P421 T422 A423 I424 K435 V439 G441 R452 A454 A470  42 R396 E399 K402 S404 T405 — — — — — — — —  43 R405 L408 S411 S413 K414 E415 E416 R427 V431 G433 E444 A446 Q462  44 R405 L408 S411 S413 K414 E415 E416 R427 V431 G433 E444 A446 Q462  45 — — — — — — — — — — — — —  46 — — — — — — — — — — — — —  47 H290 D293 G296 P298 T299 S300 I301 K312 V316 G318 G329 A331 E347  48 H319 D322 G325 P327 T328 A329 I330 K341 V345 G347 R358 A360 G376  49 — — — — — — — — — — — — —  50 R189 N192 S195 S197 L198 D199 E200 K211 V215 G217 G228 A230 E246  51 R358 E361 L364 P366 E367 D368 R369 R380 I384 G386 E397 S399 H415  52 R358 H361 T364 D366 A367 A368 A369 S380 V384 A386 P397 A399 D415  53 R358 H361 T364 D366 A367 A368 A369 S380 V384 A386 P397 A399 D415  54 H97 D100 G103 P105 T106 S107 I108 K119 V123 G125 G136 A138 E154  55 R358 E361 L364 P366 E367 D368 R369 R380 I384 G386 E397 S399 H415  56 R357 D360 L363 D365 D366 E367 E368 D379 L383 G385 P396 A398 D414  57 R373 Q276 S379 P381 A382 D383 Q384 Q395 V399 G401 P412 A414 A430  58 R360 D363 L366 D368 D369 Q370 E371 A382 V386 G388 K399 A401 N417  59 R360 D363 K366 D368 R369 N370 K371 Q382 I386 S388 E399 A401 D417  60 R359 E362 A365 E367 R368 G369 E370 K381 V385 G387 K398 A400 D416  61 R357 T360 A363 S365 D366 A367 A368 A379 V385 G385 P396 A398 E414  62 — — — — — — — — — — — — —  63 R361 Q364 E367 E369 K370 S371 D372 Q383 I387 G389 Q400 A402 E418  64 K371 E374 Q377 D379 D380 Q381 E382 P393 I397 G399 Q410 A412 S428  65 R381 D384 F387 R389 A390 K391 E392 H403 V407 G409 K420 G422 A438  66 R367 E370 E373 P375 E376 P377 T378 R389 V393 G395 S406 A408 I424  67 R358 E361 R364 P366 N367 E368 E369 D380 L384 G386 E397 A399 D415  68 R351 E354 S357 D359 T360 D361 Q362 T373 I377 D379 P390 A392 T408  69 I368 I371 E374 S376 D377 D378 E379 A390 A394 G396 P407 A409 E425  70 R380 E383 R386 E388 L389 P390 S391 E402 V406 G408 P419 A421 E437  71 — — — — — — — — — — — — —  72 Q396 E399 R402 E404 L405 P406 S407 E418 V422 G424 P435 A437 E453  73 I363 E366 E369 S371 D372 E373 T374 Q385 I389 G391 R402 A404 V420  74 T363 K366 Q369 F371 D372 E373 D374 E385 V389 G391 E402 A404 R420  75 T363 K366 Q369 F371 D372 E373 D374 E385 V389 G391 E402 A404 R420  76 R362 E365 D368 P370 Q371 N372 E373 D384 I388 G390 P401 S403 S419  77 I364 E367 E370 S372 E373 E374 A375 E386 I390 G392 H403 A405 E421  78 R361 E364 L367 P369 D370 E371 E372 R383 I387 H389 E400 G402 F418  79 R360 H363 L366 G368 E369 E370 E371 R382 I386 H388 P399 G401 F417  80 T380 E383 E386 D388 D389 D390 Q391 Q402 A406 V409 R420 A422 D438  81 T363 K366 Q369 F371 D372 E373 D374 E385 V389 G391 E402 A404 R420  82 R360 E363 A366 P368 Q369 D370 R371 A382 L386 G388 P399 A401 K417  83 R362 E365 D368 P370 Q371 N372 E373 D384 I388 G390 P401 S403 S419  84 T378 P390 A393 S395 P396 E397 E398 Q409 T413 A416 P427 A429 Q445  85 Q349 A352 Q355 S357 E358 T359 A360 R371 I375 A378 D389 A391 D407  86 R347 E350 Q353 E355 E356 R357 S358 Q369 I373 A375 Q387 A389 E405  87 T367 K370 Q373 F375 D376 E377 D378 S389 V393 G395 A406 A408 E424  88 R356 E359 D362 T364 D365 E366 E367 T378 K384 T386 S397 A399 D415  89 Q341 D344 Q347 T349 D350 E351 E352 Q363 I367 G369 E380 S382 E398  90 Q370 E373 K376 S378 D379 A380 E381 A392 I396 A398 P409 A411 Q427  91 T390 P402 A405 S407 P408 D409 E410 Q421 T425 P427 P439 A441 Q457  92 T372 E375 N378 D380 N381 E382 Q383 R394 K398 D400 K412 A414 N430  93 M386 E389 E392 S394 D395 D396 E397 D408 V412 G414 N425 S427 E443  94 Q372 E375 R378 S380 D381 A382 E383 T394 I398 E400 E411 A413 E429  95 Q351 D353 G356 D358 D359 D360 D361 P372 I376 G378 P389 A391 D407  96 Q355 E358 N361 T363 D364 E365 E366 T377 K383 T385 S396 A398 D414  97 R361 E364 L367 S369 E370 E371 E372 R383 I387 H389 E400 G402 F418  98 T430 G433 S436 S438 E439 G440 E441 K452 S458 T460 P471 A473 K489  99 R412 E415 K418 S420 T421 D422 E423 R434 I438 G440 N451 A453 D469 100 T433 G436 S439 T441 E442 G443 E444 K455 N461 K463 P474 A476 K492 101 R398 E401 K404 S406 T407 D408 E409 Q420 V434 G426 S437 A439 E455 102 T429 G432 S435 T437 E438 S439 Q440 K451 K457 G459 P470 A472 K488 103 R393 E396 N399 S401 R402 D403 E404 Q415 A419 G421 S432 A434 E450 104 T459 G462 D465 T467 Q468 D469 E470 R481 S487 K489 P500 A502 K518 105 T425 G428 S431 T433 E434 S435 D436 K447 T453 P455 P466 A468 K484 106 G405 D408 G411 P413 T414 A415 I416 K427 V431 G433 K444 A446 G462 107 T433 G436 S439 T441 E442 G443 E444 K455 N461 K463 P474 A476 K492 108 K454 G457 N460 S462 K463 D464 E465 R476 D482 K484 P495 A497 K513 109 T424 G427 S430 T432 E433 S434 D435 K446 R452 A454 P465 A467 K483 110 H450 D453 G456 P458 T459 A460 I461 K472 V476 G478 R489 A491 G507 111 T425 G428 S431 T433 E434 S435 D436 K447 R453 A455 P466 A468 K484 112 R394 E397 K400 S402 R403 T404 E405 Q416 A420 G422 S433 A435 D451 113 T440 G443 S446 T448 E449 S450 D451 K462 R468 A470 P481 A483 K499 114 T433 G436 S439 T441 E442 G443 E444 K455 N461 K463 P474 A476 K492 115 R398 K401 K404 S406 T407 D408 E409 Q420 V424 G426 S437 A439 E455 116 K454 G457 N460 S462 K463 D464 E465 R476 D482 K484 P495 A497 K513 117 R399 E402 K405 S407 T408 D409 E410 R421 V425 G427 N438 A440 D456 129 H443 D446 G449 P451 T452 S453 I454 K465 V469 G471 R482 A484 G500

In another embodiment, said differences at these amino acid positions (corresponding to the respective positions in SEQ ID NO: 2 or 4) include, but are not limited to, one or more of the following:

the amino acid corresponding to position P40 is deleted;

the amino acid corresponding to position K250 is deleted;

the amino acid corresponding to position Q391 is inserted

the amino acid corresponding to position Y520 is deleted;

the amino acid corresponding to position L521 is deleted;

the amino acid corresponding to position D522 is deleted;

the amino acid corresponding to position S523 is deleted;

the amino acid corresponding to position H524 is deleted;

the amino acid corresponding to position I525 is deleted;

the amino acid corresponding to position Y526 is deleted;

the amino acid corresponding to position V527 is deleted;

the amino acid corresponding to position K528 is deleted;

the amino acid corresponding to position M529 is deleted;

the amino acid corresponding to position D530 is deleted;

the amino acid corresponding to position E531 is deleted;

the amino acid corresponding to position K532 is deleted;

the amino acid corresponding to position T533 is deleted;

the amino acid corresponding to position A534 is deleted;

Corresponding insertions and deletion positions in other species can be found in the following table

SEQ ID Pos 1 Pos 2 Pos 3 Pos 4 Pos 5 Pos 6 Pos 7 Pos 8 Pos 9 1 P40 K250 H391 Y520 L521 D522 S523 H524 I525 2 P40 K250 H391 Y520 L521 D522 S523 H524 I525 3 P40 K249 H390 Y519 L520 D521 S522 H523 I524 4 P40 K249 H390 Y519 L520 D521 S522 H523 I524 5 P40 K250 H392 Y521 L522 D523 S524 H525 L526 7 V36 K249 H391 Y520 L521 E522 S523 — — 8 S9 K222 Q367 Y496 L497 N498 S499 S500 S501 9 P11 K221 Q366 Y495 L496 E497 S498 S499 S500 10 Q10 N220 Q364 Y493 L494 E495 S496 S497 H498 11 R11 K220 K361 Y490 L491 N492 S493 A494 S495 12 — K215 K360 Y489 L490 E491 S492 P493 S494 13 R11 K220 Q361 Y490 L491 N492 N493 A494 S495 14 R11 K217 E362 Y491 L492 E493 S494 S495 S496 15 R11 K220 Q364 Y493 L494 E495 N496 S497 S498 16 G11 K220 Q364 Y493 L494 E495 N496 S497 S498 17 R11 K220 Q361 Y490 L491 N492 N493 A494 S495 18 R10 S220 Q362 Y491 L492 E493 S494 S495 T496 19 Q5 S215 Q357 Y486 L487 E488 S489 S490 T491 20 S11 K218 Q363 Y492 L493 E494 S495 V496 S497 21 — K215 K360 Y489 L490 E491 A492 V493 S494 22 E12 K222 K365 Y494 L495 E496 S497 C498 S499 23 K12 K226 K369 Y498 L499 E500 S501 C502 S503 24 E12 K219 K358 Y487 L488 E489 S490 C491 S492 25 E58 K265 K404 Y533 L534 E535 S536 Q537 S538 26 — K191 E330 Y459 L460 D461 S462 Y463 S464 27 R39 P253 0401 Y531 L532 E533 S534 Q535 T536 28 R41 P255 0403 Y533 L534 E535 S536 H537 T538 29 E12 K192 K335 Y464 L465 E466 S467 C468 S469 30 R41 P255 Q403 Y533 L534 E535 S536 H537 T538 31 R41 P255 Q403 Y533 L534 E535 S536 H537 T538 32 L16 K227 E375 Y504 I505 N506 S507 S508 V509 33 V49 N265 — F536 L537 S538 K539 Y540 A541 34 G11 K222 Q367 H466 L467 N468 S469 S470 A471 35 K74 — K427 Y556 L557 E558 S559 C560 S561 36 R45 S259 Q407 Y537 L538 E539 S540 H541 Q542 37 R8 S219 Q367 Y497 L498 E499 S500 C501 T502 39 Q10 Q220 K363 — — — — — — 40 P7 K232 K379 D505 — — — — — 41 P7 K232 K379 D505 — — — — — 42 A15 G221 K362 — — — — — — 43 A9 E225 A371 Y500 L501 Q502 G503 S504 G505 44 V9 E225 A371 Y500 L501 Q502 G503 S504 G505 45 P40 — — — — — — — — 46 P40 — — — — — — — — 47 — K109 K256 Y385 L386 E387 S388 H389 T390 48 — K138 K285 Y405 L406 E407 S408 G409 I410 49 G1 E210 — — — — — — — 50 — — G155 — — — — — — 51 — — — H453 L454 R455 E456 E457 A458 52 — — — T453 D454 E455 R456 A457 A458 53 — — — T453 D454 E455 R456 A457 T458 54 — — K63 Y192 L193 E194 S195 H196 T197 55 — — — H453 L454 R455 E456 E457 A458 56 — — — E452 K453 — — — — 57 G14 — — — — — — — — 58 G2 — — Q455 — — — — — 59 — — — Q455 F456 T457 M458 H459 N460 60 — — — R454 I455 S456 D457 — — 61 — — — H452 L453 A454 G455 A456 D457 62 Q10 — — — — — — — — 63 — — — — — — — — — 64 E9 — — N466 T467 R468 T469 E470 E471 65 E3 — — Y476 L477 K478 T479 L480 — 66 — — — S462 L463 — — — — 67 — — — D453 R454 A455 R456 S457 — 68 — — — — — — — — — 69 — G204 — Y463 L464 K465 T466 R467 S468 70 — R201 — — — — — — — 71 — — — — — — — — — 72 T16 R220 — — — — — — — 73 M1 — — A458 L459 A460 A461 H462 G463 74 A2 — — E456 D457 G458 G459 T460 P461 75 A2 — — E456 D457 G458 G459 T460 P461 76 — — — — — — — — — 77 M2 — — A459 L460 D461 A462 Q463 G464 78 — — — G456 — — — — — 79 — — — E455 — — — — — 80 M1 V216 — H476 L477 H478 D479 C480 Q481 81 A2 — — E456 D457 G458 G459 T460 P461 82 — — — D455 I456 D457 V458 — — 83 — — — — — — — — — 84 E7 — — Q484 V485 — — — — 85 — — — K443 — — — — — 86 — — — R441 A442 A443 S444 R445 S446 87 A2 — — Q460 S461 P462 G463 — — 88 M1 K201 — — — — — — — 89 — — — N434 K435 F436 — — — 90 — A209 — Y465 L466 K467 G468 R469 — 91 — D218 — V496 R497 R498 — — — 92 — K208 — Q468 L469 S470 I471 I472 N473 93 S4 K221 — D481 L482 T483 A484 R485 V486 94 — A211 — F467 L468 Q469 S470 R471 — 95 — — — Q445 R446 — — — — 96 — K200 — S452 V453 S454 — — — 97 — K198 — S456 L457 Q458 — — — 98 G49 K260 — F529 M530 S531 R532 Y533 A534 99 P12 G237 K378 Y507 L508 E509 S510 C511 S512 100 E55 K263 — F532 L533 S534 Q535 Y536 A537 101 H10 E222 Q364 Y493 L494 E495 S496 S497 H498 102 D51 K259 — F528 L529 S530 R531 G532 V533 103 K14 — E359 Y488 L489 D490 S491 Y492 S493 104 E81 K289 — F558 L559 S560 Q561 Y562 S563 105 V44 K255 — F524 L525 T526 K527 Y528 A529 106 A9 K224 K371 Y500 L501 E502 L503 G504 I505 107 E55 K263 — F532 L533 S534 Q535 Y536 A537 108 A76 K284 — F553 L554 S555 Q556 Y557 S558 109 V46 K254 — F523 L524 T525 K526 Y527 A528 110 P55 K269 K416 Y545 L546 E547 S548 G549 I550 111 V47 K255 — F524 L525 T526 K527 Y528 A529 112 E7 Q217 K360 Y489 L490 E491 A492 V493 S494 113 V62 K270 — F539 L540 T541 K542 Y543 A544 114 E55 K263 — F532 L533 S534 Q535 Y536 A537 115 H10 E222 Q364 Y493 L494 E495 S496 S497 H498 116 A76 K284 — F553 L554 S555 Q556 Y557 S558 117 K11 G224 K365 Y494 L495 E496 S497 C498 S499 129 P40 K262 0408 Y538 L539 E540 S541 G542 I543 SEQ ID Pos 10 Pos. 11 Pos 12 Pos 13 Pos 14 Pos 15 Pos 16 Pos 17 Pos 18 1 Y526 V527 K528 M529 D530 E531 K532 T533 A534 2 Y526 V527 K528 M529 D530 E531 K532 T533 A534 3 Y525 V526 K527 M528 D529 E530 K531 T532 A533 4 Y525 V526 K527 M528 D529 E530 K531 T532 A533 5 Y527 V528 K529 M530 N531 E532 K533 T534 A535 7 — N524 K525 M526 T527 E528 E529 T530 I531 8 D502 G503 K504 M505 F506 K507 E508 — — 9 D501 D502 K503 M504 L505 K506 E507 G508 P509 10 Q499 K500 L501 L502 K503 D504 — — — 11 D496 N497 T498 V499 P500 D501 K502 — — 12 D495 E496 K497 T498 R499 H500 K501 G502 — 13 D496 N497 S498 V499 — — — — — 14 D497 G498 K499 I500 L501 Q502 Q503 G504 S505 15 D499 D500 K501 M502 L503 K504 E505 G506 S507 16 D499 D500 K501 M502 L503 K504 E505 G506 S507 17 D496 N497 T498 V499 A500 D501 K502 — — 18 D497 Q498 S499 C500 A501 E502 — — — 19 D492 Q493 S494 C495 A496 E497 — — — 20 T498 D499 S500 K501 R502 H503 C504 — — 21 A495 D496 T497 K498 N499 H500 S501 — — 22 N500 D501 K502 K503 P504 N505 D506 S507 L508 23 N504 D505 K506 K507 P508 D509 E510 S511 L512 24 N493 D494 K495 K496 P497 N498 D499 S500 L501 25 N539 D540 K541 K542 P543 N544 D545 S546 L547 26 N465 Q466 K467 — — — — — — 27 K537 H538 N539 N540 S541 H542 — — — 28 K539 H540 N541 N542 L543 H544 — — — 29 N470 D471 K472 K473 P474 N475 D476 S477 — 30 K539 H540 N541 N542 S543 H544 — — — 31 K539 H540 N541 N542 S543 H544 — — — 32 — — — — — — — — — 33 R542 S543 F544 — — — — — — 34 D472 D473 K474 M475 L476 K477 K478 G479 S480 35 N562 D563 K564 K565 P566 E567 D568 S569 — 36 A543 R544 — — — — — — — 37 D503 Q504 D505 N506 — — — — — 39 — — — — — — — — — 40 — — — — — — — — — 41 — — — — — — — — — 42 — — — — — — — — — 43 G506 K507 K508 V509 F510 T511 M512 A513 S514 44 G506 K507 K508 V509 F510 T511 M512 A513 S514 45 — — — — — — — — — 46 — — — — — — — — — 47 K391 H392 N393 N394 S395 H396 — — — 48 K411 Q412 V413 S414 — — — — — 49 — — — — — — — — — 50 — — — — — — — — — 51 A459 G460 G461 L462 A463 K464 L465 V466 L467 52 0459 P460 H461 — — — — — — 53 0459 P460 H461 — — — — — — 54 K198 H199 N200 N201 S202 H203 — — — 55 A459 G460 G461 L462 A463 K464 L465 V466 L467 56 — — — — — — — — — 57 — — — — — — — — — 58 — — — — — — — — — 59 V461 Q462 — — — — — — — 60 — — — — — — — — — 61 R458 A459 V460 A461 A462 T463 G464 P465 — 62 — — — — — — — — — 63 — — — — — — — — — 64 S472 — — — — — — — — 65 — — — — — — — — — 66 — — — — — — — — — 67 — — — — — — — — — 68 — — — — — — — — — 69 A469 V470 A471 S472 L473 R474 — — — 70 — — — — — — — — — 71 — — — — — — — — — 72 — — — — — — — — — 73 T464 T465 A466 V467 S468 T469 E470 T471 A472 74 G462 — — — — — — — — 75 G462 — — — — — — — — 76 — — — — — — — — — 77 T465 T466 A467 D468 T469 L470 E471 Q472 A473 78 — — — — — — — — — 79 — — — — — — — — — 80 T482 A483 N484 — — — — — — 81 G462 — — — — — — — — 82 — — — — — — — — — 83 — — — — — — — — — 84 — — — — — — — — — 85 — — — — — — — — — 86 1447 A448 — — — — — — — 87 — — — — — — — — — 88 — — — — — — — — — 89 — — — — — — — — — 90 — — — — — — — — — 91 — — — — — — — — — 92 D474 N475 — — — — — — — 93 — — — — — — — — — 94 — — — — — — — — — 95 — — — — — — — — — 96 — — — — — — — — — 97 — — — — — — — — — 98 Y535 K536 — — — — — — — 99 N513 D514 N515 K516 P517 E518 D519 S520 L521 100 Y538 K539 — — — — — — — 101 D499 K500 L501 L502 K503 — — — — 102 Y534 K535 — — — — — — — 103 D494 E495 K496 R497 C498 — — — — 104 D564 K565 — — — — — — — 105 Y530 K531 — — — — — — — 106 K506 R507 D508 N509 — — — — — 107 Y538 K539 — — — — — — — 108 D559 K560 — — — — — — — 109 Y529 K530 — — — — — — — 110 K551 H552 V553 N554 — — — — — 111 Y530 K531 — — — — — — — 112 A495 D496 T497 K498 N499 H500 S501 — — 113 Y545 K546 — — — — — — — 114 Y538 K539 — — — — — — — 115 D499 K500 L501 L502 K503 — — — — 116 D559 K560 — — — — — — — 117 N500 D501 K502 K503 S504 E505 D506 S507 L508 129 — — K544 Q545 D546 N547 — — —

The inventors or the present invention have surprisingly found out that a substitution of amino acids at Gly210/211, Q119, S387, or S412, or at other non-active sites, in particular when combined with a substitution at positions at other non-active sites or when combined with mutations at active sites, i.e. corresponding to positions 128, 397, and/or 420 of the Amaranthus type II PPO increase herbicide tolerance.

Consequently, in preferred embodiments, combinations of mutations corresponding to the respective positions in SEQ ID NO: 2 or 4 are encompassed by the present invention and are shown in the following Table AA.

TABLE AA double Triple quadruple quintuple Q119L, R128A Q119T, G210T, S387L Q119L, R128A, Q323H, Q119T, S387L, S412N, S412N M414Q, E436I Q119L, Q323H Q119T, G211A, S387L Q119L, Q323H, L397D, S412N Q119T, S387L Q119T, G211A, F420M Q119L, Q323H, S412N, F420M Q119L, L397D Q119L, R128A, F420M Q119T, S387L, S412N, M414Q Q119L, S412N Q119T, G210T, F420M Q119T, S387L, M414Q, E436I Q119T, M414Q Q119L, Q323H, S412N R128A, G211A, S387L, F420M Q119L, F420M Q119T, S387L, S412N Q119T, E436I Q119T, S387L, M414Q K127N, R128C Q119T, S387L, F420M R128A, G210T Q119T, S387L, E436I R128A, G211A Q119L, L397D, F420M R128A, S387L Q119T, S412N, E436I R128A, S412N Q119T, M414Q, E436I D202A, S387V R128A, G210T, F420V G210C, G211A R128A, G211A, F420M G210T, S387L R128A, S412N, F420M G210C, L397Q R128A, F420V, d_H524 G210T, S412N R128A, F420V, d_V527 G210T, M414Q G210T, G211A, M414Q G210S, F420M G211A, S387L, F420M G211A, S387L G211C, L397Q, F420V G211C, L397Q S387V, S412N, M414Q G211A, M414Q S387L, S412N, E436I G211A, L397Q S387L, M414Q, E436I G211A, F420M L397D, S412N, F420M Q323H, S412N L397E, F420M, d, H524 M343T, F420M L397E, F420M, d, V527 D372E, K373D i_Q391, H392K, N393H D372E, F420M R128A, G211N, F420M S387L, L397Q R128A, G210S, F420M S387V, S412N R128A, G211C, F420M S387L, M414Q S387L, F420M S387V, R425Y S387L, E436I L397D, S412N S412N, M414Q S412N, F420M S412N, E436I M414Q, E436I M414Q, F420M F420M, d_H524 F420M, d_V527 E436I, F420M G211C, F420V G211D, F420M G211N, F420M G210T, G211C

It will be within the knowledge of the skilled artisan to identify conserved regions and motifs shared between the homologues, orthologues and paralogues. Having identified such conserved regions that may represent suitable binding motifs, amino acids can be chosen to be substituted by any other amino acid, for example by conserved amino acids, preferably by the amino acid substitutions described SUPRA using SEQ ID NO:2 or 4 as reference.

Accordingly, the plant or plant of the present invention comprises a polynucleotide encoding a mutated PPO polypeptide which comprises one or more of the following motifs.

-   -   i) Motif 1:         -   GT[C/S]GGDP (SEQ ID NO: 131)         -   Wherein the glycine at position 4, and/or 5 within said             motif of the corresponding wildtype sequence is substituted             by any other amino acid     -   ii) Motif 2:         -   [A/S/C]PS[D/N][X][X]L (SEQ ID NO: 132)         -   Wherein the serine at position 3 within said motif of the             corresponding wildtype sequence is substituted by any other             amino acid     -   iii) Motif 3:         -   [R/Q][E/D]KQQ[L/Y]P (SEQ ID NO: 133)         -   Wherein the glutamine at position 4 within said motif of the             corresponding wildtype sequence is substituted by any other             amino acid     -   iv) Motif 4:         -   L[I/V]PSKE (SEQ ID NO: 134)         -   wherein the serine at position 4 within said motif of the             corresponding wildtype sequence is substituted by any other             amino acid.

Preferably, the mutated PPO polypeptide of the present invention in addition comprises one or more of the following motifs

-   -   a. Motif 5: SQ[N/K/H]KRYI (SEQ ID NO: 135), wherein the Arg at         position 5 within said motif is substituted by any other amino         acid;     -   b. Motif 6: TLGTLFSS (SEQ ID NO: 136), wherein the Leu at         position 2 within said motif is substituted by by any other         amino acid;     -   c. Motif 7: [F/Y]TTF[V/I]GG (SEQ ID NO: 137), wherein the Phe at         position 4 within said motif is substituted by by any other         amino acid.

Another object refers to a method of identifying a nucleotide sequence encoding a mutated PPO which is resistant or tolerant to a herbicide, the method comprising:

-   a) generating a library of mutated PPO-encoding nucleic acids, -   b) screening a population of the resulting mutated PPO-encoding     nucleic acids by expressing each of said nucleic acids in a cell or     plant and treating said cell or plant with a herbicide, -   c) comparing the herbicide-tolerance levels provided by said     population of mutated PPO encoding nucleic acids with the     herbicide-tolerance level provided by a control PPO-encoding nucleic     acid, -   d) selecting at least one mutated PPO-encoding nucleic acid that     provides a significantly increased level of tolerance to a herbicide     as compared to that provided by the control PPO-encoding nucleic     acid.

Herbicide-tolerance levels may also be determined by measuring the detoxification rate in a cell, tissue, or plant.

Detoxification rate is the rate of herbicide degradation within a certain timeframe in a respective tissue. The degradation and product formation can be determined analytically for instance by liquid chromatographic (LC) coupled to a high resolution (HR) mass spectrometer (MS). Product can be determined by comparison to authentic standards and/or by structure elucidation.

In a preferred embodiment, the mutated PPO-encoding nucleic acid selected in step d) provides at least 2-fold as much resistance or tolerance of a cell or plant to a herbicide as compared to that provided by the control PPO-encoding nucleic acid.

In a further preferred embodiment, the mutated PPO-encoding nucleic acid selected in step d) provides at least 2-fold, at least 5-fold, at least 10-fold, at least 20-fold, at least 50-fold, at least 100-fold, at least 500-fold, as much resistance or tolerance of a cell or plant to a herbicide as compared to that provided by the control PPO-encoding nucleic acid.

The resistance or tolerance can be determined by generating a transgenic plant or host cell, preferably a plant cell, comprising a nucleic acid sequence of the library of step a) and comparing said transgenic plant with a control plant or host cell, preferably a plant cell.

Many methods well known to the skilled artisan are available for obtaining suitable candidate nucleic acids for identifying a nucleotide sequence encoding a mutated PPO from a variety of different potential source organisms including microbes, plants, fungi, algae, mixed cultures etc. as well as environmental sources of DNA such as soil. These methods include inter alia the preparation of cDNA or genomic DNA libraries, the use of suitably degenerate oligonucleotide primers, the use of probes based upon known sequences or complementation assays (for example, for growth upon tyrosine) as well as the use of mutagenesis and shuffling in order to provide recombined or shuffled mutated PPO-encoding sequences.

Nucleic acids comprising candidate and control PPO encoding sequences can be expressed in yeast, in a bacterial host strain, in an alga or in a higher plant such as tobacco or Arabidopsis and the relative levels of inherent tolerance of the PPO encoding sequences screened according to a visible indicator phenotype of the transformed strain or plant in the presence of different concentrations of the selected herbicide. Dose responses and relative shifts in dose responses associated with these indicator phenotypes (formation of brown color, growth inhibition, herbicidal effect etc) are conveniently expressed in terms, for example, of GR50 (concentration for 50% reduction of growth) or MIC (minimum inhibitory concentration) values where increases in values correspond to increases in inherent tolerance of the expressed PPO. For example, in a relatively rapid assay system based upon transformation of a bacterium such as E. coli, each mutated PPO encoding sequence may be expressed, for example, as a DNA sequence under expression control of a controllable promoter such as the lacZ promoter and taking suitable account, for example by the use of synthetic DNA, of such issues as codon usage in order to obtain as comparable a level of expression as possible of different PPO sequences. Such strains expressing nucleic acids comprising alternative candidate PPO sequences may be plated out on different concentrations of the selected herbicide in, optionally, a tyrosine supplemented medium and the relative levels of inherent tolerance of the expressed PPO enzymes estimated on the basis of the extent and MIC for inhibition of the formation of the brown, ochronotic pigment, or by measuring the herbicide degradation via LC-HRMS (liquid chromatography high resolution mass spectrometry).

In another embodiment, candidate nucleic acids are transformed into plant material to generate a transgenic plant, regenerated into morphologically normal fertile plants which are then measured for differential tolerance to selected herbicides as described in the Example section hereinafter. Many suitable methods for transformation using suitable selection markers such as kanamycin, binary vectors such as from Agrobacterium and plant regeneration as, for example, from tobacco leaf discs are well known in the art. Optionally, a control population of plants is likewise transformed with a nucleic acid expressing the control PPO. The average, and distribution, of herbicide tolerance levels of a range of primary plant transformation events or their progeny to herbicides described supra are evaluated in the normal manner based upon plant damage, meristematic bleaching symptoms etc. at a range of different concentrations of herbicides. These data can be expressed in terms of, for example, GR50 values derived from dose/response curves having “dose” plotted on the x-axis and “percentage kill”, “herbicidal effect”, “numbers of emerging green plants” etc. plotted on the y-axis where increased GR50 values correspond to increased levels of inherent tolerance of the expressed PPO. Herbicides can suitably be applied pre-emergence or post-emergence.

Another object of the present invention refers to an isolated and/or recombinantly produced and/or synthetic nucleic acid molecule comprising a nucleic acid molecule encoding a mutated PPO polypeptide selected from the group consisting of:

-   -   (a) a nucleic acid molecule encoding a mutated PPO polypeptide         comprising the sequence of-SEQ ID NO: 1, 2, 3, 4, 5, 6, 7, 8, 9,         10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25,         26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41,         42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57,         58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73,         74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89,         90, 91, 92, 93, 94, 95, 96, 97, 98, 99, 100, 101, 102, 103, 104,         105, 106, 107, 108, 109, 110, 111, 112, 113, 114, 115, 116, 117,         or 129, or a variant, paralogue, orthologue or homolog thereof;     -   (b) a nucleic acid molecule, which, as a result of the         degeneracy of the genetic code, can be derived from a nucleic         acid molecule encoding a mutated PPO polypeptide comprising the         sequence of-SEQ ID NO: 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12,         13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28,         29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44,         45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60,         61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76,         77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92,         93, 94, 95, 96, 97, 98, 99, 100, 101, 102, 103, 104, 105, 106,         107, 108, 109, 110, 111, 112, 113, 114, 115, 116, 117, or 129,         or a variant, paralogue, orthologue or homolog thereof and         confers increased herbicide tolerance or resistance, as compared         to a corresponding, e.g. non-transformed, wild type plant cell,         a plant or a part thereof;     -   (c) a nucleic acid molecule encoding a mutated PPO polypeptide         having 30% or more identity, preferably at least 40%, 50%, 60%,         70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, 99,5% or more,         with the amino acid sequence of the PPO polypeptide sequence of         SEQ ID NO: 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15,         16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31,         32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47,         48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63,         64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79,         80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95,         96, 97, 98, 99, 100, 101, 102, 103, 104, 105, 106, 107, 108,         109, 110, 111, 112, 113, 114, 115, 116, 117, or 129, and confers         increased herbicide tolerance or resistance, as compared to a         corresponding, e.g. non-transformed, wild type plant cell, a         plant or a part thereof;     -   (d) nucleic acid molecule which hybridizes with a nucleic acid         molecule of (a), (b), or (c), under stringent hybridization         conditions and confers increased herbicide tolerance or         resistance, as compared to a corresponding, e.g.         non-transformed, wild type plant cell, a plant or a part         thereof;     -    wherein the amino acid sequence of the encoded mutated PPO         polypeptide differs from the wildtype amino acid sequence of a         PPO polypeptide at one or more positions corresponding to         positions 32, 53, 57, 61, 63, 64, 65, 67, 71, 76, 82, 83, 85,         86, 87, 88, 91, 103, 104, 106, 108, 116, 119, 126, 127, 129,         139, 159, 210, 211, 224, 245, 246, 248, 249, 252, 253, 254, 255,         257, 259, 260, 262, 264, 286, 291, 305, 308, 309, 323, 335, 343,         345, 358, 372, 373, 387, 391, 392, 400, 412, 414, 415, 425, 428,         431, 433, 434, 435, 436, 447, 451, 453, 464, 466, 482 of SEQ ID         NO: 1 or 2, wherein said difference refers to a substitution of         the amino acid at that positions by any other amino acid.

In a preferred embodiment, the amino acid sequence of the encoded mutated PPO polypeptide additionally differs from the wildtype amino acid sequence of a PPO polypeptide at one or more positions corresponding to positions 128, 397, or 420 of SEQ ID NO: 1 or 2, wherein said difference refers to a substitution of the amino acid at that positions by any other amino acid.

In another preferred embodiment, the amino acids at positions corresponding to positions 128, 211, 387, and 420 of SEQ ID NO: 1 or 2 are substituted by any other amino acid. Preferably, the amino acid corresponding to position 128 is substituted by Ala, the amino acid corresponding to position 211 is substituted by Ala, the amino acid corresponding to position 387 is substituted by Leu, and the amino acid corresponding to position 420 is substituted by Met.

In another preferred embodiment, the amino acids at positions corresponding to positions 128, 211, and 420 of SEQ ID NO: 1 or 2 are substituted by any other amino acid. Preferably, the amino acid corresponding to position 128 is substituted by Ala, the amino acid corresponding to position 211 is substituted by Asn or Cys, and the amino acid corresponding to position 420 is substituted by Met.

In another preferred embodiment, the amino acids at positions corresponding to positions 128, 210, and 420 of SEQ ID NO: 1 or 2 are substituted by any other amino acid. Preferably, the amino acid corresponding to position 128 is substituted by Ala, the amino acid corresponding to position 210 is substituted by Ser, and the amino acid corresponding to position 420 is substituted by Met.

In another preferred embodiment, the amino acids at positions corresponding to positions 128, 412, and 420 of SEQ ID NO: 1 or 2 are substituted by any other amino acid. Preferably, the amino acid corresponding to position 128 is substituted by Ala, the amino acid corresponding to position 412 is substituted by Asn, and the amino acid corresponding to position 420 is substituted by Met.

In another preferred embodiment, the amino acids at positions corresponding to positions 119, 128, and 420 of SEQ ID NO: 1 or 2 are substituted by any other amino acid. Preferably, the amino acid corresponding to position 119 is substituted by Leu, the amino acid corresponding to position 128 is substituted by Ala, and the amino acid corresponding to position 420 is substituted by Met.

In another preferred embodiment, the amino acids at positions corresponding to positions 211, 397 and 420 of SEQ ID NO: 1 or 2 are substituted by any other amino acid. Preferably, the amino acid corresponding to position 211 is substituted by Ala, the amino acid corresponding to position 397 is substituted by Gln, and the amino acid corresponding to position 420 is substituted by Met.

In another preferred embodiment, amino acids at positions corresponding to positions 211, and 420 of SEQ ID NO: 1 or 2 are substituted by any other amino acid.

Preferably, the amino acid corresponding to position 211 is substituted by Cys, and the amino acid corresponding to position 420 is substituted by Val.

Preferably, the amino acid corresponding to position 211 is substituted by Ala, and the amino acid corresponding to position 420 is substituted by Val.

Preferably, the amino acid corresponding to position 211 is substituted by Asp, and the amino acid corresponding to position 420 is substituted by Met.

Preferably, the amino acid corresponding to position 211 is substituted by Asn, and the amino acid corresponding to position 420 is substituted by Met.

In another preferred embodiment, amino acids at positions corresponding to positions 210, and 211 of SEQ ID NO: 1 or 2 are substituted by any other amino acid. Preferably, the amino acid corresponding to position 210 is substituted by Thr, and the amino acid corresponding to position 211 is substituted by Cys.

In a preferred embodiment, the encoded mutated PPO is a variant of SEQ ID NO:2 or 4, which includes one or more of the following:

the amino acid corresponding to position G32 is substituted by any other amino acid

the amino acid corresponding to position V53 is substituted by any other amino acid

the amino acid corresponding to position A57 is substituted by any other amino acid

the amino acid corresponding to position K61 is substituted by any other amino acid

the amino acid corresponding to position K63 is substituted by any other amino acid

the amino acid corresponding to position S64 is substituted by any other amino acid

the amino acid corresponding to position H65 is substituted by any other amino acid

the amino acid corresponding to position L67 is substituted by any other amino acid

the amino acid corresponding to position L71 is substituted by any other amino acid

the amino acid corresponding to position S76 is substituted by any other amino acid

the amino acid corresponding to position L82 is substituted by any other amino acid

the amino acid corresponding to position K83 is substituted by any other amino acid

the amino acid corresponding to position V85 is substituted by any other amino acid

the amino acid corresponding to position K86 is substituted by any other amino acid

the amino acid corresponding to position K87 is substituted by any other amino acid

the amino acid corresponding to position D88 is substituted by any other amino acid

the amino acid corresponding to position I91 is substituted by any other amino acid

the amino acid corresponding to position E103 is substituted by any other amino acid

the amino acid corresponding to position A104 is substituted by any other amino acid

the amino acid corresponding to position V106 is substituted by any other amino acid

the amino acid corresponding to position S108 is substituted by any other amino acid

the amino acid corresponding to position R116 is substituted by any other amino acid

the amino acid corresponding to position Q119 is substituted by any other amino acid,

preferably Leu

the amino acid corresponding to position K127 is substituted by any other amino acid

the amino acid corresponding to position N126 is substituted by any other amino acid

the amino acid corresponding to position Y129 is substituted by any other amino acid

the amino acid corresponding to position L139 is substituted by any other amino acid

the amino acid corresponding to position Q159 is substituted by any other amino acid

the amino acid corresponding to position G210 is substituted by any other amino acid,

preferably Ser;

the amino acid corresponding to position G211 is substituted by any other amino acid,

preferably Cys, Ala, Asp, Thr, or Asn;

the amino acid corresponding to position E224 is substituted by any other amino acid

the amino acid corresponding to position L245 is substituted by any other amino acid

the amino acid corresponding to position K248 is substituted by any other amino acid

the amino acid corresponding to position S246 is substituted by any other amino acid

the amino acid corresponding to position E249 is substituted by any other amino acid

the amino acid corresponding to position G252 is substituted by any other amino acid

the amino acid corresponding to position E253 is substituted by any other amino acid

the amino acid corresponding to position N254 is substituted by any other amino acid

the amino acid corresponding to position A255 is substituted by any other amino acid

the amino acid corresponding to position I257 is substituted by any other amino acid

the amino acid corresponding to position K259 is substituted by any other amino acid

the amino acid corresponding to position P260 is substituted by any other amino acid

the amino acid corresponding to position V262 is substituted by any other amino acid

the amino acid corresponding to position G264 is substituted by any other amino acid

the amino acid corresponding to position D286 is substituted by any other amino acid

the amino acid corresponding to position Q291 is substituted by any other amino acid

the amino acid corresponding to position P305 is substituted by any other amino acid

the amino acid corresponding to position G308 is substituted by any other amino acid

the amino acid corresponding to position N309 is substituted by any other amino acid

the amino acid corresponding to position Q323 is substituted by any other amino acid

the amino acid corresponding to position R335 is substituted by any other amino acid

the amino acid corresponding to position M343 is substituted by any other amino acid

the amino acid corresponding to position F345 is substituted by any other amino acid

the amino acid corresponding to position T358 is substituted by any other amino acid

the amino acid corresponding to position D372 is substituted by any other amino acid

the amino acid corresponding to position K373 is substituted by any other amino acid

the amino acid corresponding to position S387 is substituted by any other amino acid,

preferably Leu;

the amino acid corresponding to position H391 is substituted by any other amino acid

the amino acid corresponding to position N392 is substituted by any other amino acid

the amino acid corresponding to position L400 is substituted by any other amino acid

the amino acid corresponding to position S412 is substituted by any other amino acid,

preferably Leu;

the amino acid corresponding to position M414 is substituted by any other amino acid

the amino acid corresponding to position C415 is substituted by any other amino acid

the amino acid corresponding to position R425 is substituted by any other amino acid

the amino acid corresponding to position K428 is substituted by any other amino acid

the amino acid corresponding to position N431 is substituted by any other amino acid

the amino acid corresponding to position S433 is substituted by any other amino acid

the amino acid corresponding to position M434 is substituted by any other amino acid

the amino acid corresponding to position D435 is substituted by any other amino acid

the amino acid corresponding to position E436 is substituted by any other amino acid

the amino acid corresponding to position Q447 is substituted by any other amino acid

the amino acid corresponding to position T451 is substituted by any other amino acid

the amino acid corresponding to position D453 is substituted by any other amino acid

the amino acid corresponding to position S464 is substituted by any other amino acid

the amino acid corresponding to position Q466 is substituted by any other amino acid

the amino acid corresponding to position D482 is substituted by any other amino acid

In another preferred embodiment, the encoded mutated PPO is a variant of SEQ ID NO:2 or 4, which includes one or more of the following:

the amino acid corresponding to position P40 is deleted;

the amino acid corresponding to position K250 is deleted;

the amino acid corresponding to position Q391 is inserted

the amino acid corresponding to position Y520 is deleted;

the amino acid corresponding to position L521 is deleted;

the amino acid corresponding to position D522 is deleted;

the amino acid corresponding to position S523 is deleted;

the amino acid corresponding to position H524 is deleted;

the amino acid corresponding to position I525 is deleted;

the amino acid corresponding to position Y526 is deleted;

the amino acid corresponding to position V527 is deleted;

the amino acid corresponding to position K528 is deleted;

the amino acid corresponding to position M529 is deleted;

the amino acid corresponding to position D530 is deleted;

the amino acid corresponding to position E531 is deleted;

the amino acid corresponding to position K532 is deleted;

the amino acid corresponding to position T533 is deleted;

the amino acid corresponding to position A534 is deleted;

In other aspects, the present invention encompasses a progeny or a descendant of a herbicide-tolerant plant of the present invention as well as seeds derived from the herbicide-tolerant plants of the invention and cells derived from the herbicide-tolerant plants of the invention.

In some embodiments, the present invention provides a progeny or descendant plant derived from a plant comprising in at least some of its cells a polynucleotide operably linked to a promoter operable in plant cells, the promoter capable of expressing a mutated PPO polypeptide encoded by the polynucleotide, wherein the progeny or descendant plant comprises in at least some of its cells the recombinant polynucleotide operably linked to the promoter, the expression of the mutated PPO polypeptide conferring to the progeny or descendant plant tolerance to the herbicides.

In one embodiment, seeds of the present invention preferably comprise the herbicide-tolerance characteristics of the herbicide-tolerant plant. In other embodiments, a seed is capable of germination into a plant comprising in at least some of its cells a polynucleotide operably linked to a promoter operable in plant cells, the promoter capable of expressing a mutated PPO polypeptide encoded by the polynucleotide, the expression of the mutated PPO polypeptide conferring to the progeny or descendant plant tolerance to the herbicides.

In some embodiments, plant cells of the present invention are capable of regenerating a plant or plant part. In other embodiments, plant cells are not capable of regenerating a plant or plant part. Examples of cells not capable of regenerating a plant include, but are not limited to, endosperm, seed coat (testa & pericarp), and root cap.

In another embodiment, the present invention provides a plant cell of or capable of regenerating a plant comprising in at least some of its cells a polynucleotide operably linked to a promoter operable in plant cells, the promoter capable of expressing a mutated PPO polypeptide encoded by the polynucleotide, the expression of the mutated PPO polypeptide conferring to the plant tolerance to the herbicides, wherein the plant cell comprises the recombinant polynucleotide operably linked to a promoter.

In other embodiments, the present invention provides a plant cell comprising a polynucleotide operably linked to a promoter operable in plant cells, the promoter capable of expressing a mutated PPO polypeptide encoded by the polynucleotide, the expression of the mutated PPO polypeptide conferring to the cell tolerance to the herbicides. In another embodiment, the invention refers to a plant cell transformed by a nucleic acid encoding a mutated PPO polypeptide according to the present invention, wherein expression of the nucleic acid in the plant cell results in increased resistance or tolerance to a herbicide as compared to a wild type variety of the plant cell. Preferably, the mutated PPO polypeptide encoding nucleic acid comprises a polynucleotide sequence selected from the group consisting of: a) a polynucleotide as shown in SEQ ID NO: 118, 119, 120, 121, 122, 123, 124, 125, 126, 127, 128, or 130, or a variant or derivative thereof; b) a polynucleotide encoding a polypeptide as shown in SEQ ID NO: 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, 100, 101, 102, 103, 104, 105, 106, 107, 108, 109, 110, 111, 112, 113, 114, 115, 116, 117, or 129, or a variant or derivative thereof; c) a polynucleotide comprising at least 60 consecutive nucleotides of any of a) or b); and d) a polynucleotide complementary to the polynucleotide of any of a) through c).

In some aspects, the present invention provides a plant product prepared from the herbicide-tolerant plants hereof. In some embodiments, examples of plant products include, without limitation, grain, oil, and meal. In one embodiment, a plant product is plant grain (e.g., grain suitable for use as feed or for processing), plant oil (e.g., oil suitable for use as food or biodiesel), or plant meal (e.g., meal suitable for use as feed).

In one embodiment, a plant product prepared from a plant or plant part is provided, wherein the plant or plant part comprises in at least some of its cells a polynucleotide operably linked to a promoter operable in plant cells, the promoter capable of expressing a mutated PPO polypeptide encoded by the polynucleotide, the expression of the mutated PPO polypeptide conferring to the a plant or plant part tolerance to the herbicides.

In another embodiment, the invention refers to a method of producing a transgenic plant cell with an increased resistance to a herbicide as compared to a wild type variety of the plant cell comprising, transforming the plant cell with an expression cassette comprising a polynucleotide operably linked to a promoter operable in plant cells, the promoter capable of expressing a mutated PPO polypeptide encoded by the polynucleotide.

In another embodiment, the invention refers to a method of producing a transgenic plant comprising, (a) transforming a plant cell with an expression cassette comprising a polynucleotide operably linked to a promoter operable in plant cells, the promoter capable of expressing a mutated PPO polypeptide encoded by the polynucleotide, and (b) generating a plant with an increased resistance to herbicide from the plant cell.

In some aspects, the present invention provides a method for producing a herbicide-tolerant plant. In one embodiment, the method comprises: regenerating a plant from a plant cell transformed with a polynucleotide operably linked to a promoter operable in plant cells, the promoter capable of expressing a mutated PPO polypeptide encoded by the polynucleotide, the expression of the mutated PPO polypeptide conferring to the plant tolerance to the herbicides.

The term “expression/expressing” or “gene expression” means the transcription of a specific gene or specific genes or specific genetic construct. The term “expression” or “gene expression” in particular means the transcription of a gene or genes or genetic construct into structural RNA (rRNA, tRNA) or mRNA with or without subsequent translation of the latter into a protein. The process includes transcription of DNA and processing of the resulting mRNA product.

To obtain the desired effect, i.e. plants that are tolerant or resistant to the herbicide derivative herbicide of the present invention, it will be understood that the at least one nucleic acid is “over-expressed” by methods and means known to the person skilled in the art.

The term “increased expression” or “overexpression” as used herein means any form of expression that is additional to the wild-type expression level. Methods for increasing expression of genes or gene products are well documented in the art and include, for example, overexpression driven by appropriate promoters, the use of transcription enhancers or translation enhancers. Isolated nucleic acids which serve as promoter or enhancer elements may be introduced in an appropriate position (typically upstream) of a non-heterologous form of a polynucleotide so as to upregulate expression of a nucleic acid encoding the polypeptide of interest. For example, endogenous promoters may be altered in vivo by mutation, deletion, and/or substitution (see, Kmiec, U.S. Pat. No. 5,565,350; Zarling et al., WO9322443), or isolated promoters may be introduced into a plant cell in the proper orientation and distance from a gene of the present invention so as to control the expression of the gene. If polypeptide expression is desired, it is generally desirable to include a polyadenylation region at the 3′-end of a polynucleotide coding region. The polyadenylation region can be derived from the natural gene, from a variety of other plant genes, or from T-DNA. The 3′ end sequence to be added may be derived from, for example, the nopaline synthase or octopine synthase genes, or alternatively from another plant gene, or less preferably from any other eukaryotic gene. An intron sequence may also be added to the 5′ untranslated region (UTR) or the coding sequence of the partial coding sequence to increase the amount of the mature message that accumulates in the cytosol. Inclusion of a spliceable intron in the transcription unit in both plant and animal expression constructs has been shown to increase gene expression at both the mRNA and protein levels up to 1000-fold (Buchman and Berg (1988) Mol. Cell biol. 8: 4395-4405; Callis et al. (1987) Genes Dev 1:1183-1200). Such intron enhancement of gene expression is typically greatest when placed near the 5′ end of the transcription unit. Use of the maize introns Adh1-5 intron 1, 2, and 6, the Bronze-1 intron are known in the art. For general information see: The Maize Handbook, Chapter 116, Freeling and Walbot, Eds., Springer, N.Y. (1994).

Where appropriate, nucleic acid sequences may be optimized for increased expression in a transformed plant. For example, coding sequences that comprise plant-preferred codons for improved expression in a plant can be provided. See, for example, Campbell and Gowri (1990) Plant Physiol., 92: 1-11 for a discussion of host-preferred codon usage. Methods also are known in the art for preparing plant-preferred genes. See, for example, U.S. Pat. Nos. 5,380,831, and 5,436,391, and Murray et al. (1989) Nucleic Acids Res. 17:477-498, herein incorporated by reference.

Consequently, mutated PPO nucleic acids of the invention are provided in expression cassettes for expression in the plant of interest. The cassette will include regulatory sequences operably linked to a mutated PPO nucleic acid sequence of the invention. The term “regulatory element” as used herein refers to a polynucleotide that is capable of regulating the transcription of an operably linked polynucleotide. It includes, but not limited to, promoters, enhancers, introns, 5′ UTRs, and 3′ UTRs. By “operably linked” is intended a functional linkage between a promoter and a second sequence, wherein the promoter sequence initiates and mediates transcription of the DNA sequence corresponding to the second sequence. Generally, operably linked means that the nucleic acid sequences being linked are contiguous and, where necessary to join two protein coding regions, contiguous and in the same reading frame. The cassette may additionally contain at least one additional gene to be cotransformed into the organism. Alternatively, the additional gene(s) can be provided on multiple expression cassettes.

Such an expression cassette is provided with a plurality of restriction sites for insertion of the mutated PPO nucleic acid sequence to be under the transcriptional regulation of the regulatory regions. The expression cassette may additionally contain selectable marker genes. The expression cassette of the present invention will include in the 5′-3′ direction of transcription, a transcriptional and translational initiation region (i.e., a promoter), a mutated PPO encoding nucleic acid sequence of the invention, and a transcriptional and translational termination region (i.e., termination region) functional in plants. The promoter may be native or analogous, or foreign or heterologous, to the plant host and/or to the mutated PPO nucleic acid sequence of the invention. Additionally, the promoter may be the natural sequence or alternatively a synthetic sequence. Where the promoter is “foreign” or “heterologous” to the plant host, it is intended that the promoter is not found in the native plant into which the promoter is introduced. Where the promoter is “foreign” or “heterologous” to the mutated PPO nucleic acid sequence of the invention, it is intended that the promoter is not the native or naturally occurring promoter for the operably linked mutated TriA nucleic acid sequence of the invention. As used herein, a chimeric gene comprises a coding sequence operably linked to a transcription initiation region that is heterologous to the coding sequence. While it may be preferable to express the mutated PPO nucleic acids of the invention using heterologous promoters, the native promoter sequences may be used. Such constructs would change expression levels of the mutated PPO protein in the plant or plant cell. Thus, the phenotype of the plant or plant cell is altered.

The termination region may be native with the transcriptional initiation region, may be native with the operably linked mutated PPO sequence of interest, may be native with the plant host, or may be derived from another source (i.e., foreign or heterologous to the promoter, the mutated PPO nucleic acid sequence of interest, the plant host, or any combination thereof). Convenient termination regions are available from the Ti-plasmid of A. tumefaciens, such as the octopine synthase and nopaline synthase termination regions. See also Guerineau et al. (1991) Mol. Gen. Genet. 262: 141-144; Proudfoot (1991) Cell 64:671-674; Sanfacon et al. (1991) Genes Dev. 5: 141-149; Mogen et al. (1990) Plant Cell 2: 1261-1272; Munroe et al. (1990) Gene 91: 151-158; Ballas t al. (1989) Nucleic Acids Res. 17:7891-7903; and Joshi et al. (1987) Nucleic Acid Res. 15:9627-9639. Where appropriate, the gene(s) may be optimized for increased expression in the transformed plant. That is, the genes can be synthesized using plant-preferred codons for improved expression. See, for example, Campbell and Gowri (1990) Plant Physiol. 92: 1-11 for a discussion of host-preferred codon usage. Methods are available in the art for synthesizing plant-preferred genes. See, for example, U.S. Pat. Nos. 5,380,831, and 5,436,391, and Murray et al. (1989) Nucleic Acids Res. 17:477-498, herein incorporated by reference.

Thus, the present invention provides an expression cassette comprising a mutated PPO nucleic acid nucleic acid molecule according to the present invention and a promoter operable in plant cells.

While the polynucleotides of the invention may find use as selectable marker genes for plant transformation, the expression cassettes of the invention can include another selectable marker gene for the selection of transformed cells. Selectable marker genes, including those of the present invention, are utilized for the selection of transformed cells or tissues. Marker genes include, but are not limited to, genes encoding antibiotic resistance, such as those encoding neomycin phosphotransferase II (NEO) and hygromycin phosphotransferase (HPT), as well as genes conferring resistance to herbicidal compounds, such as glufosinate ammonium, bromoxynil, imidazolinones, and 2,4-dichlorophenoxyacetate (2,4-D). See generally, Yarranton (1992) Curr. Opin. Biotech. 3:506-511; Christophers on et al (1992) Proc. Natl. Acad. ScL USA 89:6314-6318; Yao et al. (1992) Cell 71:63-72; Reznikoff (1992) Mol Microbiol 6:2419-2422; Barkley et al (1980) in The Operon, pp. 177-220; Hu et al (1987) Cell 48:555-566; Brown et al (1987) Cell 49:603-612; Figge et al (1988) Cell 52:713-722; Deuschle et al (1989) Proc. Natl Acad. AcL USA 86:5400-5404; Fuerst et al (1989) Proc. Natl Acad. ScL USA 86:2549-2553; Deuschle et al (1990) Science 248:480-483; Gossen (1993) Ph.D. Thesis, University of Heidelberg; Reines et al (1993) Proc. Natl Acad. ScL USA 90: 1917-1921; Labow et al (1990) Mol Cell Biol 10:3343-3356; Zambretti et al (1992) Proc. Natl Acad. ScL USA 89:3952-3956; Bairn et al (1991) Proc. Natl Acad. ScL USA 88:5072-5076; Wyborski et al (1991) Nucleic Acids Res. 19:4647-4653; Hillenand-Wissman (1989) Topics Mol Struc. Biol 10: 143-162; Degenkolb et al (1991) Antimicrob. Agents Chemother. 35: 1591-1595; Kleinschnidt et al (1988) Biochemistry 27: 1094-1104; Bonin (1993) Ph.D. Thesis, University of Heidelberg; Gossen et al (1992) Proc. Natl Acad. ScL USA 89:5547-5551; Oliva et al (1992) Antimicrob. Agents Chemother. 36:913-919; Hlavka et al (1985) Handbook of Experimental Pharmacology, Vol. 78 (Springer-Verlag, Berlin); Gill et al (1988) Nature 334:721-724. Such disclosures are herein incorporated by reference. The above list of selectable marker genes is not meant to be limiting. Any selectable marker gene can be used in the present invention.

Further, additional sequence modifications are known to enhance gene expression in a cellular host. These include elimination of sequences encoding spurious polyadenylation signals, exon-intron splice site signals, transposon-like repeats, and other such well—characterized sequences that may be deleterious to gene expression. The G-C content of the sequence may be adjusted to levels average for a given cellular host, as calculated by reference to known genes expressed in the host cell. Also, if desired, sequences can be readily modified to avoid predicted hairpin secondary mRNA structures. Nucleotide sequences for enhancing gene expression can also be used in the plant expression vectors. These include, for example, introns of the maize Adh gene Adh1-S intron 1, 2, and 6 (Callis et al. Genes and Development 1: 1183-1200, 1987), and leader sequences, (W-sequence) from the Tobacco Mosaic virus (TMV), Maize Chlorotic Mottle Virus and Alfalfa Mosaic Virus (Gallie et al. Nucleic Acid Res. 15:8693-8711, 1987 and Skuzeski et al. Plant Mol. Biol. 15:65-79, 1990). The first intron from the shrunken-1 locus of maize has been shown to increase expression of genes in chimeric gene constructs. U.S. Pat. Nos. 5,424,412 and 5,593,874 disclose the use of specific introns in gene expression constructs, and Gallie et al. (Plant Physiol. 106:929-939, 1994) also have shown that introns are useful for regulating gene expression on a tissue specific basis. To further enhance or to optimize gene expression, the plant expression vectors of the invention also may contain DNA sequences containing matrix attachment regions (MARs). Plant cells transformed with such modified expression systems, then, may exhibit overexpression or constitutive expression of a nucleotide sequence of the invention.

The invention further provides an isolated recombinant expression vector comprising the expression cassette containing a mutated PPO nucleic acid nucleic acid as described above, wherein expression of the vector in a host cell results in increased tolerance to a herbicide as compared to a wild type variety of the host cell. As used herein, the term “vector” refers to a nucleic acid molecule capable of transporting another nucleic acid to which it has been linked. One type of vector is a “plasmid,” which refers to a circular double stranded DNA loop into which additional DNA segments can be ligated. Another type of vector is a viral vector, wherein additional DNA segments can be ligated into the viral genome. Certain vectors are capable of autonomous replication in a host cell into which they are introduced (e.g., bacterial vectors having a bacterial origin of replication and episomal mammalian vectors). Other vectors (e.g., non-episomal mammalian vectors) are integrated into the genome of a host cell upon introduction into the host cell, and thereby are replicated along with the host genome. Moreover, certain vectors are capable of directing the expression of genes to which they are operatively linked. Such vectors are referred to herein as “expression vectors.” In general, expression vectors of utility in recombinant DNA techniques are often in the form of plasmids. In the present specification, “plasmid” and “vector” can be used interchangeably as the plasmid is the most commonly used form of vector. However, the invention is intended to include such other forms of expression vectors, such as viral vectors (e.g., replication defective retroviruses, adenoviruses, and adeno-associated viruses), which serve equivalent functions.

The recombinant expression vectors of the invention comprise a nucleic acid of the invention in a form suitable for expression of the nucleic acid in a host cell, which means that the recombinant expression vectors include one or more regulatory sequences, selected on the basis of the host cells to be used for expression, which is operably linked to the nucleic acid sequence to be expressed. Regulatory sequences include those that direct constitutive expression of a nucleotide sequence in many types of host cells and those that direct expression of the nucleotide sequence only in certain host cells or under certain conditions. It will be appreciated by those skilled in the art that the design of the expression vector can depend on such factors as the choice of the host cell to be transformed, the level of expression of polypeptide desired, etc. The expression vectors of the invention can be introduced into host cells to thereby produce polypeptides or peptides, including fusion polypeptides or peptides, encoded by nucleic acids as described herein (e.g., mutated PPO polypeptides, fusion polypeptides, etc.)

Expression vectors may additionally contain 5′ leader sequences in the expression construct. Such leader sequences can act to enhance translation. Translation leaders are known in the art and include: picornavirus leaders, for example, EMCV leader (Encephalomyo carditis 5′ noncoding region) (Elroy-Stein et al. (1989) PNAS, 86:6126-6130); potyvirus leaders, for example, TEV leader (Tobacco Etch Virus) (Gallie et al. (1995) Gene 165(2):233-238), MDMV leader (Maize Dwarf Mosaic Virus) (Virology 154:9-20), and human immunoglobulin heavy-chain binding protein (BiP) (Macejak et al. (1991) Nature 353:90-94); untranslated leader from the coat protein mRNA of alfalfa mosaic virus (AMV RNA 4) (Jobling et al. (1987) Nature 325:622-625); tobacco mosaic virus leader (TMV) (Gallie et al. (1989) in Molecular Biology of RNA, ed. Cech (Liss, New York), pp. 237-256); and maize chlorotic mottle virus leader (MCMV) (Lommel et al. (1991) Virology 81:382-385). See also, Della-Cioppa et al. (1987) Plant Physiol. 84:965-968.

Other methods known to enhance translation also can be utilized, for example, introns, and the like. In preparing an expression vector, the various nucleic acid fragments may be manipulated, so as to provide for the nucleic acid sequences in the proper orientation and, as appropriate, in the proper reading frame. Toward this end, adapters or linkers may be employed to join the nucleic acid fragments or other manipulations may be involved to provide for convenient restriction sites, removal of superfluous nucleic acid, removal of restriction sites, or the like. For this purpose, in vitro mutagenesis, primer repair, restriction, annealing, resubstitutions, e.g., transitions and transversions, may be involved.

A number of promoters can be used in the practice of the invention. The promoters can be selected based on the desired outcome. The nucleic acids can be combined with constitutive, tissue-preferred, or other promoters for expression in plants.

Constitutive promoters include, for example, the core promoter of the Rsyn7 promoter and other constitutive promoters disclosed in WO 99/43838 and U.S. Pat. No. 6,072,050; the core CaMV 35S promoter (Odell et al. (1985) Nature 313:810-812); rice actin (McElroy et al. (1990) Plant Cell 2: 163-171); ubiquitin (Christensen et al. (1989) Plant Mol. Biol. 12:619-632 and Christensen et al. (1992) Plant Mol. Biol. 18:675-689); pEMU (Last et al. (1991) Theor. Appl. Genet. 81:581-588); MAS (Velten et al. (1984) EMBO J. 3:2723-2730); ALS promoter (U.S. Pat. No. 5,659,026), and the like. Other constitutive promoters include, for example, U.S. Pat. Nos. 5,608,149; 5,608, 144; 5,604,121; 5,569,597; 5,466,785; 5,399,680; 5,268,463; 5,608,142; and 6,177,611.

Tissue-preferred promoters can be utilized to target enhanced expression within a particular plant tissue. Such tissue-preferred promoters include, but are not limited to, leaf-preferred promoters, root-preferred promoters, seed-preferred promoters, and stem-preferred promoters. Some examples of tissue-preferred promoters are described by, e.g., Yamamoto et al. (1997) Plant J. 12(2):255-265; Kawamata et al. (1997) Plant Cell Physiol. 38(7):792-803; Hansen et al. (1997) Mol. Gen Genet. 254(3):337-343; Russell et al. (1997) Transgenic Res. 6(2): 157-168; Rinehart et al. (1996) Plant Physiol. 1 12(3): 1331-1341; Van Camp et al. (1996) Plant Physiol. 112(2):525-535; Canevascini et al. (1996) Plant Physiol. 1 12(2):513-524; Yamamoto et al. (1994) Plant Cell Physiol. 35(5):773-778; Lam (1994) Results Probl. Cell Differ. 20:181-196; Orozco of al. (1993) Plant Mol Biol. 23(6): 1 129-1138; Matsuoka et al. (1993) Voc Natl. Acad. ScL USA 90(20):9586-9590; and Guevara-Garcia et al. (1993) Plant J 4(3):495-505. Promoters can be modified, if necessary, for weak expression.

In some embodiments, the nucleic acids of interest can be targeted to the chloroplast for expression. In this manner, where the nucleic acid of interest is not directly inserted into the chloroplast, the expression vector will additionally contain a chloroplast-targeting sequence comprising a nucleotide sequence that encodes a chloroplast transit peptide to direct the gene product of interest to the chloroplasts. Such transit peptides are known in the art. With respect to chloroplast-targeting sequences, “operably linked” means that the nucleic acid sequence encoding a transit peptide (i.e., the chloroplast-targeting sequence) is linked to the desired coding sequence of the invention such that the two sequences are contiguous and in the same reading frame. See, for example, Von Heijne et al. (1991) Plant Mol. Biol. Rep. 9: 104-126; Clark et al. (1989) J Biol. Chem. 264:17544-17550; Della-Cioppa et al. (1987) Plant Physiol. 84:965-968; Romer et al. (1993) Biochem. Biophys. Res. Commun. 196: 1414-1421; and Shah et al. (1986) Science 233:478-481. For example, a chloroplast transit peptide known in the art can be fused to the amino acid sequence of a PPO polypeptide of the invention by operably linking a choloroplast-targeting sequence to the 5′-end of a nucleotide sequence encoding the PPO polypeptide.

Chloroplast targeting sequences are known in the art and include the chloroplast small subunit of ribulose-1,5-bisphosphate carboxylase (Rubisco) (de Castro Silva Filho et al. (1996) Plant Mol. Biol. 30:769-780; Schnell et al. (1991) J Biol. Chem. 266(5):3335-3342); EPSPS (Archer et al. (1990) J Bioenerg. Biomemb. 22(6):789-810); tryptophan synthase (Zhao et al. (1995) J Biol. Chem. 270(11):6081-6087); plastocyanin (Lawrence et al. (1997) J Biol. Chem. 272(33):20357-20363); chorismate synthase (Schmidt et al. (1993) J Biol. Chem. 268(36):27447-27457); and the light harvesting chlorophyll a/b binding protein (LHBP) (Lamppa et al. (1988) J Biol. Chem. 263: 14996-14999). See also Von Heijne et al. (1991) Plant Mol. Biol. Rep. 9: 104-126; Clark et al. (1989) J Biol. Chem. 264: 17544-17550; Della-Cioppa et al. (1987) Plant Physiol. 84:965-968; Romer et al. (1993) Biochem Biophys. Res. Commun. 196: 1414-1421; and Shah et al. (1986) Science 233:478-481.

Methods for transformation of chloroplasts are known in the art. See, for example, Svab et al. (1990) Proc. Natl. Acad. ScL USA 87:8526-8530; Svab and Maliga (1993) Proc. Natl. Acad. Sci. USA 90:913-917; Svab and Maliga (1993) EMBO J. 12:601-606. The method relies on particle gun delivery of DNA containing a selectable marker and targeting of the DNA to the plastid genome through homologous recombination. Additionally, plastid transformation can be accomplished by transactivation of a silent plastid-borne transgene by tissue-preferred expression of a nuclear-encoded and plastid-directed RNA polymerase. Such a system has been reported in McBride et al. (1994) Proc. Natl. Acad. Sci. USA 91:7301-7305.

The nucleic acids of interest to be targeted to the chloroplast may be optimized for expression in the chloroplast to account for differences in codon usage between the plant nucleus and this organelle. In this manner, the nucleic acids of interest may be synthesized using chloroplast-preferred codons. See, for example, U.S. Pat. No. 5,380,831, herein incorporated by reference.

Numerous plant transformation vectors and methods for transforming plants are available. See, for example, An, G. et al. (1986) Plant PysioL, 81:301-305; Fry, J., et al. (1987) Plant Cell Rep. 6:321-325; Block, M. (1988) Theor. Appl. Genet .16: 161-1 1 A; Hinchee, et al. (1990) Stadler. Genet. Symp. 2032

2.203-2

2; Cousins, et al. (1991) Aust. J. Plant Physiol. 18:481-494; Chee, P. P. and Slightom, J. L. (1992) Gene. I I 8:255-260; Christou, et al. (1992) Trends. Biotechnol. 10:239-246; Halluin, et al. (1992) Bio/Technol. 10:309-314; Dhir, et al. (1992) Plant Physiol. 99:81-88; Casas et al. (1993) Proc. Nat. Acad Sd. USA 90: 1 1212-1 1216; Christou, P. (1993) In Vitro Cell. Dev. Biol.-Plant; 29P.119-124; Davies, et al. (1993) Plant Cell Rep. 12: 180-183; Dong, J. A. and Mchughen, A. (1993) Plant ScL 91: 139-148; Franklin, C. I. and Trieu, T. N. (1993) Plant. Physiol. 102: 167; Golovkin, et al. (1993) Plant ScL 90:41-52; Guo Chin ScL Bull. 38:2072-2078; Asano, et al. (1994) Plant Cell Rep. 13; Ayeres N. M. and Park, W. D. (1994) Crit. Rev. Plant. Sci. 13:219-239; Barcelo, et al. (1994) Plant. J. 5:583-592; Becker, et al. (1994) Plant. J. 5:299-307; Borkowska et al. (1994) Acta. Physiol Plant. 16:225-230; Christou, P. (1994) Agro. Food. Ind. Hi Tech. 5: 17-27; Eapen et al. (1994) Plant Cell Rep. 13:582-586; Hartman, et al. (1994) Bio-Technology 12: 919923; Ritala, et al. (1994) Plant. Mol. Biol. 24:317-325; and Wan, Y. C. and Lemaux, P. G. (1994) Plant Physiol. 104:3748.

In some embodiments, the methods of the invention involve introducing a polynucleotide construct into a plant. By “introducing” is intended presenting to the plant the polynucleotide construct in such a manner that the construct gains access to the interior of a cell of the plant. The methods of the invention do not depend on a particular method for introducing a polynucleotide construct to a plant, only that the polynucleotide construct gains access to the interior of at least one cell of the plant. Methods for introducing polynucleotide constructs into plants are known in the art including, but not limited to, stable transformation methods, transient transformation methods, and virus-mediated methods. The term “introduction” or “transformation” as referred to herein further means the transfer of an exogenous polynucleotide into a host cell, irrespective of the method used for transfer. Plant tissue capable of subsequent clonal propagation, whether by organogenesis or embryogenesis, may be transformed with a genetic construct of the present invention and a whole plant regenerated there from. The particular tissue chosen will vary depending on the clonal propagation systems available for, and best suited to, the particular species being transformed. Exemplary tissue targets include leaf disks, pollen, embryos, cotyledons, hypocotyls, megagametophytes, callus tissue, existing meristematic tissue (e.g., apical meristem, axillary buds, and root meristems), and induced meristem tissue (e.g., cotyledon meristem and hypocotyl meristem). The polynucleotide may be transiently or stably introduced into a host cell and may be maintained non-integrated, for example, as a plasmid. Alternatively, it may be integrated into the host genome. The resulting transformed plant cell may then be used to regenerate a transformed plant in a manner known to persons skilled in the art.

By “stable transformation” is intended that the polynucleotide construct introduced into a plant integrates into the genome of the plant and is capable of being inherited by descendent thereof. By “transient transformation” is intended that a polynucleotide construct introduced into a plant does not integrate into the genome of the plant.

For the transformation of plants and plant cells, the nucleotide sequences of the invention are inserted using standard techniques into any vector known in the art that is suitable for expression of the nucleotide sequences in a plant or plant cell. The selection of the vector depends on the preferred transformation technique and the target plant species to be transformed. In an embodiment of the invention, the encoding nucleotide sequence is operably linked to a plant promoter, e.g. a promoter known in the art for high-level expression in a plant cell, and this construct is then introduced into a plant cell that is susceptible to herbicides; and a transformed plant is regenerated. In some embodiments, the transformed plant is tolerant to exposure to a level of herbicides that would kill or significantly injure a plant regenerated from an untransformed cell. This method can be applied to any plant species or crops.

Methodologies for constructing plant expression vectors and introducing foreign nucleic acids into plants are generally known in the art. For example, foreign DNA can be introduced into plants, using tumor-inducing (Ti) plasmid vectors. Other methods utilized for foreign DNA delivery involve the use of PEG mediated protoplast transformation, electroporation, microinjection whiskers, and biolistics or microprojectile bombardment for direct DNA uptake. Such methods are known in the art. (U.S. Pat. No. 5,405,765 to Vasil et al.; Bilang et a (1991) Gene 100: 247-250; Scheid et al. al, (1991) MoL Gen. Genet., 228: 104-1 12; Guerche et al., (1987) Plant Science 52:111-116; Neuhause et al., (1987) Theor. Appl Genet. 75: 30-36; Klein et al., (1987) Nature 327: 70-73; Howell et al., (1980) Science 208: 1265; Horsch et al., (1985) Science 227: 1229-1231; DeBlock et al., (1989) Plant Physiology 91: 694-701; Methods for Plant Molecular Biology (Weissbach and Weissbach, eds.) Academic Press, Inc. (1988) and Methods in Plant Molecular Biology (Schuler and Zielinski, eds.) Academic Press, Inc. (1989).

Other suitable methods of introducing nucleotide sequences into plant cells include microinjection as described by, e.g., Crossway et al. (1986) Biotechniques 4:320-334, electroporation as described by e.g., Riggs et al. (1986) Proc. Natl. Acad. ScL USA 83:5602-5606, Agrobacterium-mediated transformation as described by e.g., Townsend et al., U.S. Pat. No. 5,563,055, Zhao et al., U.S. Pat. No. 5,981,840, direct gene transfer as described by, e.g., Paszkowski et al. (1984) EMBO J. 3:2717-2722, and ballistic particle acceleration as described by, e.g., U.S. Pat. Nos. 4,945,050; 5,879,918; 5,886,244; and 5,932,782; Tomes et al. (1995) “Direct DNA Transfer into Intact Plant Cells via Microprojectile Bombardment,” in Plant Cell, Tissue, and Organ Culture: Fundamental Methods, ed. Gamborg and Phillips (Springer-Verlag, Berlin); McCabe et al. (1988) Biotechnology 6:923-926); and Led transformation (WO 00/28058). Also see, Weissinger et al., (1988) Ann. Rev. Genet. 22:421-477; Sanford et al, (1987) Particulate Science and Technology 5:27-37 (onion); Christou et al, (1988) Plant Physiol. 87:671-674 (soybean); McCabe et al., (1988) Bio/Technology 6:923-926 (soybean); Finer and McMullen (1991) In Vitro Cell Dev. Biol. 27P: 175-182 (soybean); Singh et al, (1998) Theor. Appl. Genet. 96:319-324 (soybean); Datta et al., (1990) Biotechnology 8:736-740 (rice); Klein et al., (1988) PNAS, 85:4305-4309 (maize); Klein et al., (1988) Biotechnology 6:559-563 (maize); U.S. Pat. Nos. 5,240,855; 5,322,783; and 5,324,646; Tomes et al., (1995) “Direct DNA Transfer into Intact Plant Cells via Microprojectile Bombardment,” in Plant Cell, Tissue, and Organ Culture: Fundamental Methods, ed. Gamborg (Springer-Verlag, Berlin) (maize); Klein et al., (1988) Plant Physiol. 91:440-444 (maize); Fromm et al., (1990) Biotechnology 8:833-839 (maize); Hooykaas-Van Slogteren et al., (1984) Nature (London) 311:763-764; Bowen et al, U.S. Pat. No. 5,736,369 (cereals); Bytebier et al, (1987) PNAS 84:5345-5349 (Liliaceae); De Wet et al., (1985) in The Experimental Manipulation of Ovule Tissues, ed. Chapman et al, (Longman, New York), pp. 197-209 (pollen); Kaeppler et al., (1990) Plant Cell Reports 9:415-418 and Kaeppler et al., (1992) Theor. Apph Genet. 84:560-566 (whisker-mediated transformation); D′Halluin et al., (1992) Plant Cell 4: 1495-1505 (electroporation); Li et al., (1993) Plant Cell Reports 12:250-255 and Christou and Ford (1995) Annals of Botany 75:407-413 (rice); Osjoda et al, (1996) Nature Biotechnology 14:745-750 (maize via Agrobacterium tumefaciens); each of which is herein incorporated by reference.

Transgenic plants, including transgenic crop plants, are preferably produced via Agrobacterium-mediated transformation. An advantageous transformation method is the transformation in planta. To this end, it is possible, for example, to allow the agrobacteria to act on plant seeds or to inoculate the plant meristem with agrobacteria. It has proved particularly expedient in accordance with the invention to allow a suspension of transformed agrobacteria to act on the intact plant or at least on the flower primordia. The plant is subsequently grown on until the seeds of the treated plant are obtained (Clough and Bent, Plant J. (1998) 16, 735-743). Methods for Agrobacterium-mediated transformation of rice include well known methods for rice transformation, such as those described in any of the following: European patent application EP 1198985 A1, Aldemita and Hodges (Planta 199: 612-617, 1996); Chan et al. (Plant Mol Biol 22 (3): 491-506, 1993), Hiei et al. (Plant J 6 (2): 271-282, 1994), which disclosures are incorporated by reference herein as if fully set forth. In the case of corn transformation, the preferred method is as described in either Ishida et al. (Nat. Biotechnol 14(6): 745-50, 1996) or Frame et al. (Plant Physiol 129(1): 13-22, 2002), which disclosures are incorporated by reference herein as if fully set forth. Said methods are further described by way of example in B. Jenes et al., Techniques for Gene Transfer, in: Transgenic Plants, Vol. 1, Engineering and Utilization, eds. S. D. Kung and R. Wu, Academic Press (1993) 128-143 and in Potrykus Annu. Rev. Plant Physiol. Plant Molec. Biol. 42 (1991) 205-225). The nucleic acids or the construct to be expressed is preferably cloned into a vector, which is suitable for transforming Agrobacterium tumefaciens, for example pBin19 (Bevan et al., Nucl. Acids Res. 12 (1984) 8711). Agrobacteria transformed by such a vector can then be used in known manner for the transformation of plants, such as plants used as a model, like Arabidopsis (Arabidopsis thaliana is within the scope of the present invention not considered as a crop plant), or crop plants such as, by way of example, tobacco plants, for example by immersing bruised leaves or chopped leaves in an agrobacterial solution and then culturing them in suitable media. The transformation of plants by means of Agrobacterium tumefaciens is described, for example, by Höfgen and Willmitzer in Nucl. Acid Res. (1988) 16, 9877 or is known inter alia from F. F. White, Vectors for Gene Transfer in Higher Plants; in Transgenic Plants, Vol. 1, Engineering and Utilization, eds. S. D. Kung and R. Wu, Academic Press, 1993, pp. 15-38.

One transformation method known to those of skill in the art is the dipping of a flowering plant into an Agrobacteria solution, wherein the Agrobacteria contains the PPO nucleic acid, followed by breeding of the transformed gametes. Agrobacterium mediated plant transformation can be performed using for example the GV3101(pMP90) (Koncz and Schell, 1986, Mol. Gen. Genet. 204:383-396) or LBA4404 (Clontech) Agrobacterium tumefaciens strain. Transformation can be performed by standard transformation and regeneration techniques (Deblaere et al., 1994, Nucl. Acids. Res. 13:4777-4788; Gelvin, Stanton B. and Schilperoort, Robert A, Plant Molecular Biology Manual, 2nd Ed.—Dordrecht: Kluwer Academic Publ., 1995.—in Sect., Ringbuc Zentrale Signatur: BT11-P ISBN 0-7923-2731-4; Glick, Bernard R. and Thompson, John E., Methods in Plant Molecular Biology and Biotechnology, Boca Raton: CRC Press, 1993 360 S., ISBN 0-8493-5164-2). For example, rapeseed can be transformed via cotyledon or hypocotyl transformation (Moloney et al., 1989, Plant Cell Report 8:238-242; De Block et al., 1989, Plant Physiol. 91:694-701). Use of antibiotics for Agrobacterium and plant selection depends on the binary vector and the Agrobacterium strain used for transformation. Rapeseed selection is normally performed using kanamycin as selectable plant marker. Agrobacterium mediated gene transfer to flax can be performed using, for example, a technique described by Mlynarova et al., 1994, Plant Cell Report 13:282-285. Additionally, transformation of soybean can be performed using for example a technique described in European Patent No. 0424 047, U.S. Pat. No. 5,322,783, European Pat. No. 0397 687, U.S. Pat. No. 5,376,543, or U.S. Pat. No. 5,169,770. Transformation of maize can be achieved by particle bombardment, polyethylene glycol mediated DNA uptake, or via the silicon carbide fiber technique. (See, for example, Freeling and Walbot “The maize handbook” Springer Verlag: New York (1993) ISBN 3-540-97826-7). A specific example of maize transformation is found in U.S. Patent No. 5,990,387, and a specific example of wheat transformation can be found in PCT Application No. WO 93/07256.

In some embodiments, polynucleotides of the present invention may be introduced into plants by contacting plants with a virus or viral nucleic acids. Generally, such methods involve incorporating a polynucleotide construct of the invention within a viral DNA or RNA molecule. It is recognized that the polypeptides of the invention may be initially synthesized as part of a viral polyprotein, which later may be processed by proteolysis in vivo or in vitro to produce the desired recombinant polypeptide. Further, it is recognized that promoters of the invention also encompass promoters utilized for transcription by viral RNA polymerases. Methods for introducing polynucleotide constructs into plants and expressing a protein encoded therein, involving viral DNA or RNA molecules, are known in the art. See, for example, U.S. Pat. Nos. 5,889,191, 5,889,190, 5,866,785, 5,589,367 and 5,316,931; herein incorporated by reference. The cells that have been transformed may be grown into plants in accordance with conventional ways. See, for example, McCormick et a (1986) Plant Cell Reports 5:81-84. These plants may then be grown, and either pollinated with the same transformed strain or different strains, and the resulting hybrid having constitutive expression of the desired phenotypic characteristic identified. Two or more generations may be grown to ensure that expression of the desired phenotypic characteristic is stably maintained and inherited and then seeds harvested to ensure expression of the desired phenotypic characteristic has been achieved.

The present invention may be used for transformation of any plant species, including, but not limited to, monocots and dicots. Examples of plant species of interest include, but are not limited to, corn or maize (Zea mays), Brassica sp. (e.g., B. napus, B. rapa, B. juncea), particularly those Brassica species useful as sources of seed oil, alfalfa (Medicago sativa), rice (Oryza sativa), rye (Secale cereale), sorghum (Sorghum bicolor, Sorghum vulgare), millet e.g., pearl millet (Pennisetum glaucum), proso millet (Panicum miliaceum), foxtail millet (Setaria italica), finger millet (Eleusine coracana)), sunflower (Helianthus annu), saffiower (Carthamus tinctorius), wheat (Triticum aestivum, T. Turgidum ssp. durum), soybean (Glycine max), tobacco (Nicotiana tabacum), potato (Solarium tuberosum), peanuts (Arachis hypogaea), cotton (Gossypium barbadense, Gossypium hirsutum), sweet potato (Ipomoea batatus), cassava (Manihot esculenta), coffee (Coffea spp.), coconut (Cocos nucifera), pineapple (Ananas comosus), citrus trees (Citrus spp.), cocoa (Theobroma cacao), tea (Camellia sinensis), banana (Musa spp.), avocado (Persea americana), fig (Ficus casica), guava (Psidium guajava), mango (Mangifera indica), olive (Olea europaea), papaya (Carica papaya), cashew (Anacardium occidentale), macadamia (Macadamia integrifolia), almond (Prunus amygdalus), sugar beets (Beta vulgaris), sugarcane (Saccharum spp.), oats, barley, vegetables, ornamentals, and conifers. Preferably, plants of the present invention are crop plants (for example, sunflower, Brassica sp., cotton, sugar, beet, soybean, peanut, alfalfa, safflower, tobacco, corn, rice, wheat, rye, barley triticale, sorghum, millet, etc.).

In addition to the transformation of somatic cells, which then have to be regenerated into intact plants, it is also possible to transform the cells of plant meristems and in particular those cells which develop into gametes. In this case, the transformed gametes follow the natural plant development, giving rise to transgenic plants. Thus, for example, seeds of Arabidopsis are treated with agrobacteria and seeds are obtained from the developing plants of which a certain proportion is transformed and thus transgenic [Feldman, K A and Marks MD (1987). Mol Gen Genet 208:274-289; Feldmann K (1992). In: C Koncz, N-H Chua and J Shell, eds, Methods in Arabidopsis Research. Word Scientific, Singapore, pp. 274-289]. Alternative methods are based on the repeated removal of the inflorescences and incubation of the excision site in the center of the rosette with transformed agrobacteria, whereby transformed seeds can likewise be obtained at a later point in time (Chang (1994). Plant J. 5: 551-558; Katavic (1994). Mol Gen Genet, 245: 363-370). However, an especially effective method is the vacuum infiltration method with its modifications such as the “floral dip” method. In the case of vacuum infiltration of Arabidopsis, intact plants under reduced pressure are treated with an agrobacterial suspension [Bechthold, N (1993). C R Acad Sci Paris Life Sci, 316: 1194-1199], while in the case of the “floral dip” method the developing floral tissue is incubated briefly with a surfactant-treated agrobacterial suspension [Clough, S J and Bent A F (1998) The Plant J. 16, 735-743]. A certain proportion of transgenic seeds are harvested in both cases, and these seeds can be distinguished from non-transgenic seeds by growing under the above-described selective conditions. In addition the stable transformation of plastids is of advantages because plastids are inherited maternally is most crops reducing or eliminating the risk of transgene flow through pollen. The transformation of the chloroplast genome is generally achieved by a process which has been schematically displayed in Klaus et al., 2004 [Nature Biotechnology 22 (2), 225-229]. Briefly the sequences to be transformed are cloned together with a selectable marker gene between flanking sequences homologous to the chloroplast genome. These homologous flanking sequences direct site specific integration into the plastome. Plastidal transformation has been described for many different plant species and an overview is given in Bock (2001) Transgenic plastids in basic research and plant biotechnology. J Mol Biol. 2001 Sep. 21; 312 (3):425-38 or Maliga, P (2003) Progress towards commercialization of plastid transformation technology. Trends Biotechnol. 21, 20-28. Further biotechnological progress has recently been reported in form of marker free plastid transformants, which can be produced by a transient co-integrated maker gene (Klaus et al., 2004, Nature Biotechnology 22(2), 225-229). The genetically modified plant cells can be regenerated via all methods with which the skilled worker is familiar. Suitable methods can be found in the abovementioned publications by S. D. Kung and R. Wu, Potrykus or Hofgen and Willmitzer.

Generally after transformation, plant cells or cell groupings are selected for the presence of one or more markers which are encoded by plant-expressible genes co-transferred with the gene of interest, following which the transformed material is regenerated into a whole plant. To select transformed plants, the plant material obtained in the transformation is, as a rule, subjected to selective conditions so that transformed plants can be distinguished from untransformed plants. For example, the seeds obtained in the above-described manner can be planted and, after an initial growing period, subjected to a suitable selection by spraying. A further possibility consists in growing the seeds, if appropriate after sterilization, on agar plates using a suitable selection agent so that only the transformed seeds can grow into plants. Alternatively, the transformed plants are screened for the presence of a selectable marker such as the ones described above.

Following DNA transfer and regeneration, putatively transformed plants may also be evaluated, for instance using Southern analysis, for the presence of the gene of interest, copy number and/or genomic organisation. Alternatively or additionally, expression levels of the newly introduced DNA may be monitored using Northern and/or Western analysis, both techniques being well known to persons having ordinary skill in the art.

The generated transformed plants may be propagated by a variety of means, such as by clonal propagation or classical breeding techniques. For example, a first generation (or T1) transformed plant may be selfed and homozygous second-generation (or T2) transformants selected, and the T2 plants may then further be propagated through classical breeding techniques. The generated transformed organisms may take a variety of forms. For example, they may be chimeras of transformed cells and non-transformed cells; clonal transformants (e.g., all cells transformed to contain the expression cassette); grafts of transformed and untransformed tissues (e.g., in plants, a transformed rootstock grafted to an untransformed scion).

Preferably, the expression of the nucleic acid in the plant results in the plant's increased resistance to herbicide as compared to a wild type variety of the plant.

In another embodiment, the invention refers to a plant, comprising a plant cell according to the present invention, wherein expression of the nucleic acid in the plant results in the plant's increased resistance to herbicide as compared to a wild type variety of the plant.

The plants described herein can be either transgenic crop plants or non-transgenic plants.

In addition to the general definition, give SUPRA, “transgenic”, “transgene” or “recombinant” means with regard to, for example, a nucleic acid sequence, an expression cassette, gene construct or a vector comprising the nucleic acid sequence or an organism transformed with the nucleic acid sequences, expression cassettes or vectors according to the invention, all those constructions brought about by recombinant methods in which either

-   (a) the nucleic acid sequences encoding proteins useful in the     methods of the invention, or -   (b) genetic control sequence(s) which is operably linked with the     nucleic acid sequence according to the invention, for example a     promoter, or -   (c) a) and b)

are not located in their natural genetic environment or have been modified by recombinant methods, it being possible for the modification to take the form of, for example, a substitution, addition, deletion, inversion or insertion of one or more nucleotide residues in order to allow for the expression of the mutated PPO of the present invention. The natural genetic environment is understood as meaning the natural genomic or chromosomal locus in the original plant or the presence in a genomic library. In the case of a genomic library, the natural genetic environment of the nucleic acid sequence is preferably retained, at least in part. The environment flanks the nucleic acid sequence at least on one side and has a sequence length of at least 50 bp, preferably at least 500 bp, especially preferably at least 1000 bp, most preferably at least 5000 bp. A naturally occurring expression cassette—for example the naturally occurring combination of the natural promoter of the nucleic acid sequences with the corresponding nucleic acid sequence encoding a polypeptide useful in the methods of the present invention, as defined above—becomes a transgenic expression cassette when this expression cassette is modified by non-natural, synthetic (“artificial”) methods such as, for example, mutagenic treatment. Suitable methods are described, for example, in U.S. Pat. No. 5,565,350 or WO 00/15815.

A transgenic plant for the purposes of the invention is thus understood as meaning, as above, that the nucleic acids of the invention are not at their natural locus in the genome of said plant, it being possible for the nucleic acids to be expressed homologously or heterologously. However, as mentioned, transgenic also means that, while the nucleic acids according to the invention or used in the inventive method are at their natural position in the genome of a plant, the sequence has been modified with regard to the natural sequence, and/or that the regulatory sequences of the natural sequences have been modified. Transgenic is preferably understood as meaning the expression of the nucleic acids according to the invention at an unnatural locus in the genome, i.e. homologous or, preferably, heterologous expression of the nucleic acids takes place. Preferred transgenic plants are mentioned herein. Furthermore, the term “transgenic” refers to any plant, plant cell, callus, plant tissue, or plant part, that contains all or part of at least one recombinant polynucleotide. In many cases, all or part of the recombinant polynucleotide is stably integrated into a chromosome or stable extra-chromosomal element, so that it is passed on to successive generations. For the purposes of the invention, the term “recombinant polynucleotide” refers to a polynucleotide that has been altered, rearranged, or modified by genetic engineering. Examples include any cloned polynucleotide, or polynucleotides, that are linked or joined to heterologous sequences. The term “recombinant” does not refer to alterations of polynucleotides that result from naturally occurring events, such as spontaneous mutations, or from non-spontaneous mutagenesis followed by selective breeding.

Plants containing mutations arising due to non-spontaneous mutagenesis and selective breeding are referred to herein as non-transgenic plants and are included in the present invention. In embodiments wherein the plant is transgenic and comprises multiple mutated PPO nucleic acids, the nucleic acids can be derived from different genomes or from the same genome. Alternatively, in embodiments wherein the plant is non-transgenic and comprises multiple mutated PPO nucleic acids, the nucleic acids are located on different genomes or on the same genome.

In certain embodiments, the present invention involves herbidicide-resistant plants that are produced by mutation breeding. Such plants comprise a polynucleotide encoding a mutated PPO and are tolerant to one or more PPO-inhibiting herbicides. Such methods can involve, for example, exposing the plants or seeds to a mutagen, particularly a chemical mutagen such as, for example, ethyl methanesulfonate (EMS) and selecting for plants that have enhanced tolerance to at least one or more PPO-inhibiting herbicide [see Example 1]. However, the present invention is not limited to herbicide-tolerant plants that are produced by a mutagenesis method involving the chemical mutagen EMS. Any mutagenesis method known in the art may be used to produce the herbicide-resistant plants of the present invention. Such mutagenesis methods can involve, for example, the use of any one or more of the following mutagens: radiation, such as X-rays, Gamma rays (e.g., cobalt 60 or cesium 137), neutrons, (e.g., product of nuclear fission by uranium 235 in an atomic reactor), Beta radiation (e.g., emitted from radioisotopes such as phosphorus 32 or carbon 14), and ultraviolet radiation (preferably from 250 to 290 nm), and chemical mutagens such as base analogues (e.g., 5-bromo-uracil), related compounds (e.g., 8-ethoxy caffeine), antibiotics (e.g., streptonigrin), alkylating agents (e.g., sulfur mustards, nitrogen mustards, epoxides, ethylenamines, sulfates, sulfonates, sulfones, lactones), azide, hydroxylamine, nitrous acid, or acridines. Herbicide-resistant plants can also be produced by using tissue culture methods to select for plant cells comprising herbicide-resistance mutations and then regenerating herbicide-resistant plants therefrom. See, for example, U.S. Pat. Nos. 5,773,702 and 5,859,348, both of which are herein incorporated in their entirety by reference. Further details of mutation breeding can be found in “Principals of Cultivar Development” Fehr, 1993 Macmillan Publishing Company the disclosure of which is incorporated herein by reference

Alternatively, herbicide-resistant plants according to the present invention can also be produced by using genome editing methods to select for plant cells comprising herbicide-resistance mutations and then regenerating herbicide-resistant plants therefrom. “Genome Editing” refers to a type of genetic engineering in which DNA is inserted, deleted or replaced in the genome of an organism using engineered nucleases. These nucleases are known to the skilled artisan to create site-specific double-strand breaks at desired locations in the genome. The induced double-strand breaks are repaired through nonhomologous end-joining or homologous recombination, resulting in targeted mutations. Known in the art are currently four families of engineered nucleases which can be used for the purposes of the present invention: meganucleases, zinc finger nucleases (ZFNs), transcription activator-like effector-based nucleases (TALEN), and the CRISPR-Cas system.-For references, see, for example, Esvelt, K M. and Wang, H H. (2013) “Genome-scale engineering for systems and synthetic biology”, Mol Syst Biol. 9 (1): 641; Tan, W S. et al., (2012) “Precision editing of large animal genomes”, Adv Genet. 80: 37-97; Puchta, H. and Fauser, F. (2013) “Gene targeting in plants: 25 years later”, Int. J. Dev. Biol. 57: 629-637; Boglioli, Elsy and Richard, Magali “Rewriting the book of life: a new era in precision genome editing”, Boston Consulting Group, Retrieved Nov. 30, 2015; Method of the Year 2011. Nat Meth 9 (1), 1-1.

The plant of the present invention comprises at least one mutated PPO nucleic acid or over-expressed wild-type PPO nucleic acid, and has increased tolerance to a PPO-inhibiting herbicide as compared to a wild-type variety of the plant. It is possible for the plants of the present invention to have multiple mutated PPO nucleic acids from different genomes since these plants can contain more than one genome. For example, a plant contains two genomes, usually referred to as the A and B genomes. Because PPO is a required metabolic enzyme, it is assumed that each genome has at least one gene coding for the PPO enzyme (i.e. at least one PPO gene). As used herein, the term “PPO gene locus” refers to the position of a PPO gene on a genome, and the terms “PPO gene” and “PPO nucleic acid” refer to a nucleic acid encoding the PPO enzyme. The PPO nucleic acid on each genome differs in its nucleotide sequence from a PPO nucleic acid on another genome. One of skill in the art can determine the genome of origin of each PPO nucleic acid through genetic crossing and/or either sequencing methods or exonuclease digestion methods known to those of skill in the art.

The present invention includes plants comprising one, two, three, or more mutated PPO alleles, wherein the plant has increased tolerance to a PPO-inhibiting herbicide as compared to a wild-type variety of the plant. The mutated PPO alleles can comprise a nucleotide sequence selected from the group consisting of a polynucleotide as defined in SEQ ID NO: 118, 119, 120, 121, 122, 123, 124, 125, 126, 127, 128 or 130, or a variant or derivative thereof, a polynucleotide encoding a polypeptide as defined in SEQ ID NO: 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, 100, 101, 102, 103, 104, 105, 106, 107, 108, 109, 110, 111, 112, 113, 114, 115, 116, 117 or 129, or a variant or derivative, homologue, orthologue, paralogue thereof, a polynucleotide comprising at least 60 consecutive nucleotides of any of the aforementioned polynucleotides; and a polynucleotide complementary to any of the aforementioned polynucleotides.

“Alleles” or “allelic variants” are alternative forms of a given gene, located at the same chromosomal position. Allelic variants encompass Single Nucleotide Polymorphisms (SNPs), as well as Small Insertion/Deletion Polymorphisms (INDELs). The size of INDELs is usually less than 100 bp. SNPs and INDELs form the largest set of sequence variants in naturally occurring polymorphic strains of most organisms

The term “variety” refers to a group of plants within a species defined by the sharing of a common set of characteristics or traits accepted by those skilled in the art as sufficient to distinguish one cultivar or variety from another cultivar or variety. There is no implication in either term that all plants of any given cultivar or variety will be genetically identical at either the whole gene or molecular level or that any given plant will be homozygous at all loci. A cultivar or variety is considered “true breeding” for a particular trait if, when the true-breeding cultivar or variety is self-pollinated, all of the progeny contain the trait. The terms “breeding line” or “line” refer to a group of plants within a cultivar defined by the sharing of a common set of characteristics or traits accepted by those skilled in the art as sufficient to distinguish one breeding line or line from another breeding line or line. There is no implication in either term that all plants of any given breeding line or line will be genetically identical at either the whole gene or molecular level or that any given plant will be homozygous at all loci. A breeding line or line is considered “true breeding” for a particular trait if, when the true-breeding line or breeding line is self-pollinated, all of the progeny contain the trait. In the present invention, the trait arises from a mutation in a PPO gene of the plant or seed.

The herbicide-resistant plants of the invention that comprise polynucleotides encoding mutated PPO polypeptides also find use in methods for increasing the herbicide-resistance of a plant through conventional plant breeding involving sexual reproduction. The methods comprise crossing a first plant that is a herbicide-resistant plant of the invention to a second plant that may or may not be resistant to the same herbicide or herbicides as the first plant or may be resistant to different herbicide or herbicides than the first plant. The second plant can be any plant that is capable of producing viable progeny plants (i.e., seeds) when crossed with the first plant. Typically, but not necessarily, the first and second plants are of the same species. The methods can optionally involve selecting for progeny plants that comprise the mutated PPO polypeptides of the first plant and the herbicide resistance characteristics of the second plant. The progeny plants produced by this method of the present invention have increased resistance to a herbicide when compared to either the first or second plant or both. When the first and second plants are resistant to different herbicides, the progeny plants will have the combined herbicide tolerance characteristics of the first and second plants. The methods of the invention can further involve one or more generations of backcrossing the progeny plants of the first cross to a plant of the same line or genotype as either the first or second plant. Alternatively, the progeny of the first cross or any subsequent cross can be crossed to a third plant that is of a different line or genotype than either the first or second plant.

The present invention also provides plants, plant organs, plant tissues, plant cells, seeds, and non-human host cells that are transformed with the at least one polynucleotide molecule, expression cassette, or transformation vector of the invention. Such transformed plants, plant organs, plant tissues, plant cells, seeds, and non-human host cells have enhanced tolerance or resistance to at least one herbicide, at levels of the herbicide that kill or inhibit the growth of an untransformed plant, plant tissue, plant cell, or non-human host cell, respectively. Preferably, the transformed plants, plant tissues, plant cells, and seeds of the invention are Arabidopsis thaliana and crop plants.

In another embodiment, the invention refers to a seed produced by a transgenic plant comprising a plant cell of the present invention, wherein the seed is true breeding for an increased resistance to a herbicide as compared to a wild type variety of the seed.

In other aspects, herbicide-tolerant plants of the present invention can be employed as herbicide-tolerance trait donor lines for development, as by traditional plant breeding, to produce other varietal and/or hybrid crops containing such trait or traits. All such resulting variety or hybrids crops, containing the ancestral herbicide-tolerance trait or traits can be referred to herein as progeny or descendant of the ancestral, herbicide-tolerant line(s).

In other embodiments, the present invention provides a method for producing a herbicide-tolerant plant. The method comprises: crossing a first herbicide-tolerant plant with a second plant to produce a herbicide-tolerant progeny plant, wherein the first plant and the progeny plant comprise in at least some of their cells a polynucleotide operably linked to a promoter operable in plant cells, the recombinant polynucleotide being effective in the cells of the first plant to express a mutated PPO polypeptide encoded by the polynucleotide, the expression of the mutated PPO polypeptide conferring to the plant tolerance to herbicides.

Traditional plant breeding might be employed whereby the herbicide-tolerant trait is introduced in the progeny plant resulting therefrom. In one embodiment, the present invention provides a method for producing a herbicide-tolerant progeny plant, the method comprising: crossing a parent plant with a herbicide-tolerant plant to introduce the herbicide-tolerance characteristics of the herbicide-tolerant plant into the germplasm of the progeny plant, wherein the progeny plant has increased tolerance to the herbicides relative to the parent plant. In other embodiments, the method further comprises the step of introgressing the herbicide-tolerance characteristics through traditional plant breeding techniques to obtain a descendent plant having the herbicide-tolerance characteristics.

In other aspects, plants of the invention include those plants which, in addition to being tolerant to PPO-inhibiting herbicides, have been subjected to further genetic modifications by breeding, mutagenesis or genetic engineering, e.g. have been rendered tolerant to applications of specific other classes of herbicides, such as AHAS inhibitors; auxinic herbicides; bleaching herbicides such as hydroxyphenylpyruvate dioxygenase (HPPD) inhibitors or phytoene desaturase (PDS) inhibitors; EPSPS inhibitors such as glyphosate; glutamine synthetase (GS) inhibitors such as glufosinate; lipid biosynthesis inhibitors such as acetyl CoA carboxylase (ACCase) inhibitors; or oxynil {i.e. bromoxynil or ioxynil) herbicides as a result of conventional methods of breeding or genetic engineering, Thus, herbicide-tolerant plants of the invention can be made resistant to multiple classes of herbicides through multiple genetic modifications, such as resistance to both glyphosate and glufosinate or to both glyphosate and a herbicide from another class such as HPPD inhibitors, AHAS inhibitors, or ACCase inhibitors. These herbicide resistance technologies are, for example, described in Pest Management Science (at volume, year, page): 61, 2005, 246; 61, 2005, 258; 61, 2005, 277; 61, 2005, 269; 61, 2005, 286; 64, 2008, 326; 64, 2008, 332; Weed Science 57, 2009, 108; Australian Journal of Agricultural Research 58, 2007, 708; Science 316, 2007, 1185; and references quoted therein. For example, herbicide-tolerant plants of the invention, in some embodiments, may be tolerant to ACCase inhibitors, such as “dims” {e.g., cycloxydim, sethoxydim, clethodim, or tepraloxydim), “fops” {e.g. , clodinafop, diclofop, fluazifop, haloxyfop, or quizalofop), and “dens” (such as pinoxaden); to auxinic herbicides, such as dicamba; to EPSPS inhibitors, such as glyphosate; to cellulose biosynthesis inhibitors; and to GS inhibitors, such as glufosinate.

In addition to these classes of inhibitors, herbicide-tolerant plants of the invention may also be tolerant to herbicides having other modes of action, for example, chlorophyll/carotenoid pigment inhibitors, cell membrane disrupters, photosynthesis inhibitors, cell division inhibitors, root inhibitors, shoot inhibitors, and combinations thereof.

Such tolerance traits may be expressed, e.g.: as mutant or wildtype HPPD proteins, as mutant or wildtype PPO proteins, as mutant AHASL proteins, mutant ACCase proteins, mutant EPSPS proteins, or mutant glutamine synthetase proteins; or as mutant native, inbred, or transgenic aryloxyalkanoate dioxygenase (AAD or DHT), haloarylnitrilase (BXN), 2,2-dichloropropionic acid dehalogenase (DEH), glyphosate-N-acetyltransferase (GAT), glyphosate decarboxylase (GDC), glyphosate oxidoreductase (GOX), glutathione-S-transferase (GST), phosphinothricin acetyltransferase (PAT or bar), or CYP450s proteins having an herbicide-degrading activity. Herbicide-tolerant plants hereof can also be stacked with other traits including, but not limited to, pesticidal traits such as Bt Cry and other proteins having pesticidal activity toward coleopteran, lepidopteran, nematode, or other pests; nutrition or nutraceutical traits such as modified oil content or oil profile traits, high protein or high amino acid concentration traits, and other trait types known in the art.

Furthermore, in other embodiments, herbicide-tolerant plants are also covered which are, by the use of recombinant DNA techniques and/or by breeding and/or otherwise selected for such characteristics, rendered able to synthesize one or more insecticidal proteins, especially those known from the bacterial genus Bacillus, particularly from Bacillus thuringiensis, such as [delta]-endotoxins, e.g. CryIA(b), CryIA(c), CryIF, CryIF(a2), CryIIA(b), CryIIIA, CryIIIB(bI) or Cry9c; vegetative insecticidal proteins (VIP), e.g. VIP1, VIP2, VIP3 or VIP3A; insecticidal proteins of bacteria colonizing nematodes, e.g. Photorhabdus spp. or Xenorhabdus spp.; toxins produced by animals, such as scorpion toxins, arachnid toxins, wasp toxins, or other insect-specific neurotoxins; toxins produced by fungi, such streptomycete toxins; plant lectins, such as pea or barley lectins; agglutinins; proteinase inhibitors, such as trypsin inhibitors, serine protease inhibitors, patatin, cystatin or papain inhibitors; ribosome-inactivating proteins (RIP), such as ricin, maize-RIP, abrin, luffin, saporin or bryodin; steroid metabolism enzymes, such as 3-hydroxy-steroid oxidase, ecdysteroid-IDP-glycosyl-transferase, cholesterol oxidases, ecdysone inhibitors or HMG-CoA-reductase; ion channel blockers, such as blockers of sodium or calcium channels; juvenile hormone esterase; diuretic hormone receptors (helicokinin receptors); stilben synthase, bibenzyl synthase, chitinases or glucanases. In the context of the present invention these insecticidal proteins or toxins are to be understood expressly also as pre-toxins, hybrid proteins, truncated or otherwise modified proteins. Hybrid proteins are characterized by a new combination of protein domains, (see, e.g. WO 02/015701). Further examples of such toxins or genetically modified plants capable of synthesizing such toxins are disclosed, e.g., in EP-A 374 753, WO 93/007278, WO 95/34656, EP-A 427 529, EP-A 451 878, WO 03/18810 and WO 03/52073. The methods for producing such genetically modified plants are generally known to the person skilled in the art and are described, e.g. in the publications mentioned above. These insecticidal proteins contained in the genetically modified plants impart to the plants producing these proteins tolerance to harmful pests from all taxonomic groups of arthropods, especially to beetles (Coeloptera), two-winged insects (Diptera), and moths (Lepidoptera) and to nematodes (Nematoda).

In some embodiments, expression of one or more protein toxins (e.g., insecticidal proteins) in the herbicide-tolerant plants is effective for controlling organisms that include, for example, members of the classes and orders: Coleoptera such as the American bean weevil Acanthoscelides obtectus; the leaf beetle Agelastica alni; click beetles (Agriotes lineatus, Agriotes obscurus, Agriotes bicolor); the grain beetle Ahasverus advena; the summer schafer Amphimallon solstitialis; the furniture beetle Anobium punctatum; Anthonomus spp. (weevils); the Pygmy mangold beetle Atomaria linearis; carpet beetles (Anthrenus spp., Attagenus spp.); the cowpea weevil Callosobruchus maculates; the fried fruit beetle Carpophilus hemipterus; the cabbage seedpod weevil Ceutorhynchus assimilis; the rape winter stem weevil Ceutorhynchus picitarsis; the wireworms Conoderus vespertinus and Conoderus falli; the banana weevil Cosmopolites sordidus; the New Zealand grass grub Costelytra zealandica; the June beetle Cotinis nitida; the sunflower stem weevil Cylindrocopturus adspersus; the larder beetle Dermestes lardarius; the corn rootworms Diabrotica virgifera, Diabrotica virgifera virgifera, and Diabrotica barberi; the Mexican bean beetle Epilachna varivestis; the old house borer Hylotropes bajulus; the lucerne weevil Hypera postica; the shiny spider beetle Gibbium psylloides; the cigarette beetle Lasioderma serricorne; the Colorado potato beetle Leptinotarsa decemlineata; Lyctus beetles {Lyctus spp., the pollen beetle Meligethes aeneus; the common cockshafer Melolontha melolontha; the American spider beetle Mezium americanum; the golden spider beetle Niptus hololeucs; the grain beetles Oryzaephilus surinamensis and Oryzaephilus Mercator; the black vine weevil Otiorhynchus sulcatus; the mustard beetle Phaedon cochleariae, the crucifer flea beetle Phyllotreta cruciferae; the striped flea beetle Phyllotreta striolata; the cabbage steam flea beetle Psylliodes chrysocephala; Ptinus spp. (spider beetles); the lesser grain borer Rhizopertha dominica; the pea and been weevil Sitona lineatus; the rice and granary beetles Sitophilus oryzae and Sitophilus granaries; the red sunflower seed weevil Smicronyx fulvus; the drugstore beetle Stegobium paniceum; the yellow mealworm beetle Tenebrio molitor, the flour beetles Tribolium castaneum and Tribolium confusum; warehouse and cabinet beetles {Trogoderma spp.); the sunflower beetle Zygogramma exclamationis; Dermaptera (earwigs) such as the European earwig Forficula auricularia and the striped earwig Labidura riparia; Dictyoptera such as the oriental cockroach Blatta orientalis; the greenhouse millipede Oxidus gracilis; the beet fly Pegomyia betae; the frit fly Oscinella frit; fruitflies (Dacus spp., Drosophila spp.); Isoptera (termites) including species from the familes Hodotermitidae, Kalotermitidae, Mastotermitidae, Rhinotermitidae, Serritermitidae, Termitidae, Termopsidae; the tarnished plant bug Lygus lineolaris; the black bean aphid Aphis fabae; the cotton or melon aphid Aphis gossypii; the green apple aphid Aphis pomi; the citrus spiny whitefly Aleurocanthus spiniferus; the sweet potato whitefly Bemesia tabaci; the cabbage aphid Brevicoryne brassicae; the pear psylla Cacopsylla pyricola; the currant aphid Cryptomyzus ribis; the grape phylloxera Daktulosphaira vitifoliae; the citrus psylla Diaphorina citri; the potato leafhopper Empoasca fabae; the bean leafhopper Empoasca Solana; the vine leafhopper Empoasca vitis; the woolly aphid Eriosoma lanigerum; the European fruit scale Eulecanium corni; the mealy plum aphid Hyalopterus arundinis; the small brown planthopper Laodelphax striatellus; the potato aphid Macrosiphum euphorbiae; the green peach aphid Myzus persicae; the green rice leafhopper Nephotettix cinticeps; the brown planthopper Nilaparvata lugens; the hop aphid Phorodon humuli; the bird-cherry aphid Rhopalosiphum padi; the grain aphid Sitobion avenae; Lepidoptera such as Adoxophyes orana (summer fruit tortrix moth); Archips podana (fruit tree tortrix moth); Bucculatrix pyrivorella (pear leafminer); Bucculatrix thurberiella (cotton leaf perforator); Bupalus piniarius (pine looper); Carpocapsa pomonella (codling moth); Chilo suppressalis (striped rice borer); Choristoneura fumiferana (eastern spruce budworm); Cochylis hospes (banded sunflower moth); Diatraea grandiosella (southwestern corn borer); Eupoecilia ambiguella (European grape berry moth); Helicoverpa armigera (cotton bollworm); Helicoverpa zea (cotton bollworm); Heliothis vires cens (tobacco budworm), Homeosoma electellum (sunflower moth); Homona magnanima (oriental tea tree tortrix moth); Lithocolletis blancardella (spotted tentiform leafminer); Lymantria dispar (gypsy moth); Malacosoma neustria (tent caterpillar); Mamestra brassicae (cabbage armyworm); Mamestra configurata (Bertha armyworm); Operophtera brumata (winter moth); Ostrinia nubilalis (European corn borer), Panolis flammea (pine beauty moth), Phyllocnistis citrella (citrus leafminer); Pieris brassicae (cabbage white butterfly); Rachiplusia ni (soybean looper); Spodoptera exigua (beet armywonn); Spodoptera littoralis (cotton leafworm); Sylepta derogata (cotton leaf roller); Trichoplusia ni (cabbage looper); Orthoptera such as the common cricket Acheta domesticus, tree locusts (Anacridium spp.), the migratory locust Locusta migratoria, the twostriped grasshopper Melanoplus bivittatus, the differential grasshopper Melanoplus differ entialis, the redlegged grasshopper Melanoplus femurrubrum, the migratory grasshopper Melanoplus sanguinipes, the northern mole cricket Neocurtilla hexadectyla, the red locust Nomadacris septemfasciata, the shortwinged mole cricket Scapteriscus abbreviatus, the southern mole cricket Scapteriscus borellii, the tawny mole cricket Scapteriscus vicinus, and the desert locust Schistocerca gregaria; Symphyla such as the garden symphylan Scutigerella immaculata; Thysanoptera such as the tobacco thrips Frankliniella fusca, the flower thrips Frankliniella intonsa, the western flower thrips Frankliniella occidentalism the cotton bud thrips Frankliniella schultzei, the banded greenhouse thrips Hercinothrips femoralis, the soybean thrips Neohydatothrips variabilis, Kelly's citrus thrips Pezothrips kellyanus, the avocado thrips Scirtothrips perseae, the melon thrips Thrips palmi, and the onion thrips Thrips tabaci; and the like, and combinations comprising one or more of the foregoing organisms.

In some embodiments, expression of one or more protein toxins (e.g., insecticidal proteins) in the herbicide-tolerant plants is effective for controlling flea beetles, i.e. members of the flea beetle tribe of family Chrysomelidae, preferably against Phyllotreta spp., such as Phyllotreta cruciferae and/or Phyllotreta triolata. In other embodiments, expression of one or more protein toxins {e.g., insecticidal proteins) in the herbicide-tolerant plants is effective for controlling cabbage seedpod weevil, the Bertha armyworm, Lygus bugs, or the diamondback moth.

Furthermore, in one embodiment, herbicide-tolerant plants are also covered which are, e.g. by the use of recombinant DNA techniques and/or by breeding and/or otherwise selected for such traits, rendered able to synthesize one or more proteins to increase the resistance or tolerance of those plants to bacterial, viral or fungal pathogens. The methods for producing such genetically modified plants are generally known to the person skilled in the art.

Furthermore, in another embodiment, herbicide-tolerant plants are also covered which are, e.g. by the use of recombinant DNA techniques and/or by breeding and/or otherwise selected for such traits, rendered able to synthesize one or more proteins to increase the productivity (e.g. oil content), tolerance to drought, salinity or other growth-limiting environmental factors or tolerance to pests and fungal, bacterial or viral pathogens of those plants.

Furthermore, in other embodiments, herbicide-tolerant plants are also covered which are, e.g. by the use of recombinant DNA techniques and/or by breeding and/or otherwise selected for such traits, altered to contain a modified amount of one or more substances or new substances, for example, to improve human or animal nutrition, e.g. oil crops that produce health-promoting long-chain omega-3 fatty acids or unsaturated omega-9 fatty acids (e.g. Nexera® rape, Dow Agro Sciences, Canada).

Furthermore, in some embodiments, herbicide-tolerant plants are also covered which are, e.g. by the use of recombinant DNA techniques and/or by breeding and/or otherwise selected for such traits, altered to contain increased amounts of vitamins and/or minerals, and/or improved profiles of nutraceutical compounds.

In one embodiment, herbicide-tolerant plants of the present invention, relative to a wild-type plant, comprise an increased amount of, or an improved profile of, a compound selected from the group consisting of: glucosinolates (e.g., glucoraphanin (4-methylsulfinylbutyl-glucosinolate), sulforaphane, 3-indolylmethyl-glucosinolate(glucobrassicin), 1-methoxy-3-indolylmethyl-glucosinolate (neoglucobrassicin)); phenolics (e.g., flavonoids (e.g., quercetin, kaempferol), hydroxycinnamoyl derivatives (e.g., 1,2,2′-trisinapoylgentiobiose, 1,2-diferuloylgentiobiose, 1,2′-disinapoyl-2-feruloylgentiobiose, 3-0-caffeoyl-quinic (neochlorogenic acid)); and vitamins and minerals (e.g., vitamin C, vitamin E, carotene, folic acid, niacin, riboflavin, thiamine, calcium, iron, magnesium, potassium, selenium, and zinc).

In another embodiment, herbicide-tolerant plants of the present invention, relative to a wild-type plant, comprise an increased amount of, or an improved profile of, a compound selected from the group consisting of: progoitrin; isothiocyanates; indoles (products of glucosinolate hydrolysis); glutathione; carotenoids such as beta-carotene, lycopene, and the xanthophyll carotenoids such as lutein and zeaxanthin; phenolics comprising the flavonoids such as the flavonols (e.g. quercetin, rutin), the flavans/tannins (such as the procyanidins comprising coumarin, proanthocyanidins, catechins, and anthocyanins); flavones; phytoestrogens such as coumestans, lignans, resveratrol, isoflavones e.g. genistein, daidzein, and glycitein; resorcyclic acid lactones; organosulphur compounds; phytosterols; terpenoids such as carnosol, rosmarinic acid, glycyrrhizin and saponins; chlorophyll; chlorphyllin, sugars, anthocyanins, and vanilla.

In other embodiments, herbicide-tolerant plants of the present invention, relative to a wild-type plant, comprise an increased amount of, or an improved profile of, a compound selected from the group consisting of: vincristine, vinblastine, taxanes (e.g., taxol (paclitaxel), baccatin III, 10-desacetylbaccatin III, 10-desacetyl taxol, xylosyl taxol, 7-epitaxol, 7-epibaccatin III, 10-desacetylcephalomannine, 7-epicephalomannine, taxotere, cephalomannine, xylosyl cephalomannine, taxagifine, 8-benxoyloxy taxagifine, 9-acetyloxy taxusin, 9-hydroxy taxusin, taiwanxam, taxane la, taxane Ib, taxane Ic, taxane Id, GMP paclitaxel, 9-dihydro 13-acetylbaccatin III, 10-desacetyl-7-epitaxol, tetrahydrocannabinol (THC), cannabidiol (CBD), genistein, diadzein, codeine, morphine, quinine, shikonin, ajmalacine, serpentine, and the like.

In other aspects, a method for treating a plant of the present invention is provided.

In some embodiments, the method comprises contacting the plant with an agronomically acceptable composition. In one embodiment, the agronomically acceptable composition comprises an auxinic herbicide A. I.

In another aspect, the present invention provides a method for preparing a descendent seed. The method comprises planting a seed of or capable of producing a plant of the present invention. In one embodiment, the method further comprises growing a descendent plant from the seed; and harvesting a descendant seed from the descendent plant. In other embodiments, the method further comprises applying a herbicides herbicidal composition to the descendent plant.

In another embodiment, the invention refers to harvestable parts of the transgenic plant according to the present invention. Preferably, the harvestable parts comprise the PPO nucleic acid or PPO protein of the present invention. The harvestable parts may be seeds, roots, leaves and/or flowers comprising the PPO nucleic acid or PPO protein or parts thereof. Preferred parts of soy plants are soy beans comprising the PPO nucleic acid or PPO protein.

In another embodiment, the invention refers to products derived from a transgenic plant according to the present invention, parts thereof or harvestable parts thereof. A preferred plant product is fodder, seed meal, oil, or seed-treatment-coated seeds. Preferably, the meal and/or oil comprise the PPO nucleic acids or PPO proteins.

In another embodiment, the invention refers to a method for the production of a product, which method comprises

-   a) growing the plants of the invention or obtainable by the methods     of invention and -   b) producing said product from or by the plants of the invention     and/or parts, e.g. seeds, of these plants.

In a further embodiment the method comprises the steps

-   a) growing the plants of the invention, -   b) removing the harvestable parts as defined above from the plants     and -   c) producing said product from or by the harvestable parts of the     invention.

The product may be produced at the site where the plant has been grown, the plants and/or parts thereof may be removed from the site where the plants have been grown to produce the product. Typically, the plant is grown, the desired harvestable parts are removed from the plant, if feasible in repeated cycles, and the product made from the harvestable parts of the plant. The step of growing the plant may be performed only once each time the methods of the invention is performed, while allowing repeated times the steps of product production e.g. by repeated removal of harvestable parts of the plants of the invention and if necessary further processing of these parts to arrive at the product. It is also possible that the step of growing the plants of the invention is repeated and plants or harvestable parts are stored until the production of the product is then performed once for the accumulated plants or plant parts. Also, the steps of growing the plants and producing the product may be performed with an overlap in time, even simultaneously to a large extend or sequentially. Generally the plants are grown for some time before the product is produced.

In one embodiment the products produced by said methods of the invention are plant products such as, but not limited to, a foodstuff, feedstuff, a food supplement, feed supplement, fiber, cosmetic and/or pharmaceutical. Foodstuffs are regarded as compositions used for nutrition and/or for supplementing nutrition. Animal feedstuffs and animal feed supplements, in particular, are regarded as foodstuffs.

In another embodiment the inventive methods for the production are used to make agricultural products such as, but not limited to, plant extracts, proteins, amino acids, carbohydrates, fats, oils, polymers, vitamins, and the like.

It is possible that a plant product consists of one or more agricultural products to a large extent.

Herbicides

As described above, the present invention provides nucleic acids, polypeptides, conferring tolerance of plants to herbicides.

Generally, the term “herbicide” is used herein to mean an active ingredient that kills, controls or otherwise adversely modifies the growth of plants. The preferred amount or concentration of the herbicide is an “effective amount” or “effective concentration.” By “effective amount” and “effective concentration” is intended an amount and concentration, respectively, that is sufficient to kill or inhibit the growth of a similar, wild-type, plant, plant tissue, plant cell, or host cell, but that said amount does not kill or inhibit as severely the growth of the herbicide-resistant plants, plant tissues, plant cells, and host cells of the present invention. Typically, the effective amount of a herbicide is an amount that is routinely used in agricultural production systems to kill weeds of interest. Such an amount is known to those of ordinary skill in the art. Herbicidal activity is exhibited by herbicides useful for the the present invention when they are applied directly to the plant or to the locus of the plant at any stage of growth or before planting or emergence. The effect observed depends upon the plant species to be controlled, the stage of growth of the plant, the application parameters of dilution and spray drop size, the particle size of solid components, the environmental conditions at the time of use, the specific compound employed, the specific adjuvants and carriers employed, the soil type, and the like, as well as the amount of chemical applied. These and other factors can be adjusted as is known in the art to promote non-selective or selective herbicidal action. Generally, it is preferred to apply the herbicide postemergence to relatively immature undesirable vegetation to achieve the maximum control of weeds.

By a “herbicide-tolerant” or “herbicide-resistant” plant, it is intended that a plant that is tolerant or resistant to at least one herbicide at a level that would normally kill, or inhibit the growth of, a normal or wild-type plant. By “herbicide-tolerant wildtype or mutated PPO protein” or “herbicide-resistant wildtype or mutated PPO protein”, it is intended that such a PPO protein displays higher PPO activity, relative to the PPO activity of a wild-type PPO protein, when in the presence of at least one herbicide that is known to interfere with PPO activity and at a concentration or level of the herbicide that is known to inhibit the PPO activity of the wild-type mutated PPO protein. Furthermore, the PPO activity of such a herbicide-tolerant or herbicide-resistant mutated PPO protein may be referred to herein as “herbicide-tolerant” or “herbicide-resistant” PPO activity

Examples of herbicides which can be used according to the present invention, i.e. to which the plants according to the present invention are tolerant/resistant to, are compounds known to the skilled artisan as PPO inhibiting herbicides. Examples of PPO inhibiting herbicides are described in detail hereinafter.

Generally, if the PPO-inhibiting herbicides (also referred to as compounds A) and/or the herbicidal compounds B as described herein, which can be employed in the context of the present invention, are capable of forming geometrical isomers, for example E/Z isomers, it is possible to use both, the pure isomers and mixtures thereof, in the compositions useful for the present the invention. If the PPO-inhibiting herbicides A and/or the herbicidal compounds B as described herein have one or more centers of chirality and, as a consequence, are present as enantiomers or diastereomers, it is possible to use both, the pure enantiomers and diastereomers and their mixtures, in the compositions according to the invention. If the PPO-inhibiting herbicides A and/or the herbicidal compounds B as described herein have ionizable functional groups, they can also be employed in the form of their agriculturally acceptable salts. Suitable are, in general, the salts of those cations and the acid addition salts of those acids whose cations and anions, respectively, have no adverse effect on the activity of the active compounds. Preferred cations are the ions of the alkali metals, preferably of lithium, sodium and potassium, of the alkaline earth metals, preferably of calcium and magnesium, and of the transition metals, preferably of manganese, copper, zinc and iron, further ammonium and substituted ammonium in which one to four hydrogen atoms are replaced by C₁-C₄-alkyl, hydroxy-C₁-C₄-alkyl, C₁-C₄-alkoxy-C₁-C₄-alkyl, hydroxy-C₁-C₄-alkoxy-C₁-C₄-alkyl, phenyl or benzyl, preferably ammonium, methylammonium, isopropylammonium, dimethylammonium, diisopropylammonium, trimethylammonium, heptylammonium, dodecylammonium, tetradecylammonium, tetramethylammonium, tetraethylammonium, tetrabutylammonium, 2-hydroxyethylammonium (olamine salt), 2-(2-hydroxyeth-1-oxy)eth-1-ylammonium (diglycolamine salt), di(2-hydroxyeth-1-yl)ammonium (diolamine salt), tris(2-hydroxyethyl)ammonium (trolamine salt), tris(2-hydroxypropyl)ammonium, benzyltrimethylammonium, benzyltriethylammonium, N,N,N-trimethylethanolammonium (choline salt), furthermore phosphonium ions, sulfonium ions, preferably tri(C₁-C₄-alkyl)sulfonium, such as trimethylsulfonium, and sulfoxonium ions, preferably tri(C₁-C₄-alkyl)sulfoxonium, and finally the salts of polybasic amines such as N,N-bis-(3-aminopropyl)methylamine and diethylenetriamine. Anions of useful acid addition salts are primarily chloride, bromide, fluoride, iodide, hydrogensulfate, methylsulfate, sulfate, dihydrogenphosphate, hydrogenphosphate, nitrate, bicarbonate, carbonate, hexafluorosilicate, hexafluorophosphate, benzoate and also the anions of C₁-C₄-alkanoic acids, preferably formate, acetate, propionate and butyrate.

The PPO-inhibiting herbicides A and/or the herbicidal compounds B as described herein having a carboxyl group can be employed in the form of the acid, in the form of an agriculturally suitable salt as mentioned above or else in the form of an agriculturally acceptable derivative, for example as amides, such as mono- and di-C₁-C₆-alkylamides or arylamides, as esters, for example as allyl esters, propargyl esters, C₁-C₁₀-alkyl esters, alkoxyalkyl esters, tefuryl ((tetrahydrofuran-2-yl)methyl) esters and also as thioesters, for example as C₁-C₁₀-alkylthio esters. Preferred mono- and di-C₁-C₆-alkylamides are the methyl and the dimethylamides. Preferred arylamides are, for example, the anilides and the 2-chloroanilides. Preferred alkyl esters are, for example, the methyl, ethyl, propyl, isopropyl, butyl, isobutyl, pentyl, mexyl (1-methylhexyl), meptyl (1-methylheptyl), heptyl, octyl or isooctyl (2-ethylhexyl) esters. Preferred C₁-C₄-alkoxy-C₁-C₄-alkyl esters are the straight-chain or branched C₁-C₄-alkoxy ethyl esters, for example the 2-methoxyethyl, 2-ethoxyethyl, 2-butoxyethyl (butotyl), 2-butoxypropyl or 3-butoxypropyl ester. An example of a straight-chain or branched C₁-C₁₀-alkylthio ester is the ethylthio ester.

Examples of PPO inhibiting herbicides which can be used according to the present invention are acifluorfen, acifluorfen-sodium, aclonifen, azafenidin, bencarbazone, benzfendizone, bifenox, butafenacil, carfentrazone, carfentrazone-ethyl, chlomethoxyfen, cinidon-ethyl, fluazolate, flufenpyr, flufenpyr-ethyl, flumiclorac, flumiclorac-pentyl, flumioxazin, fluoroglycofen, fluoroglycofen-ethyl, fluthiacet, fluthiacet-methyl, fomesafen, halosafen, lactofen, oxadiargyl, oxadiazon, oxyfluorfen, pentoxazone, profluazol, pyraclonil, pyraflufen, pyraflufen-ethyl, saflufenacil, sulfentrazone, thidiazimin, tiafenacil, chlornitrofen, flumipropyn, fluoronitrofen, flupropacil, furyloxyfen, nitrofluorfen, ethyl [3-[2-chloro-4-fluoro-5-(1-methyl-6-trifluoromethyl-2,4-dioxo-1,2,3,4-tetrahydropyrimidin-3-yl)phenoxy]-2-pyridyloxy]acetate (CAS 353292-31-6; S-3100), N-ethyl-3-2,6-dichloro-4-trifluoromethylphenoxy)-5-methyl-1H-pyrazole-1-carboxamide (CAS 452098-92-9), N-tetrahydrofurfuryl-3-(2,6-dichloro-4-trifluoromethylphenoxy)-5-methyl-1H-pyrazole-1-carboxamide (CAS 915396-43-9), N-ethyl-3-(2-chloro-6-fluoro-4-trifluoromethyl-phenoxy)-5-methyl-1H-pyrazole-1-carboxamide (CAS 452099-05-7), N-tetrahydrofurfuryl-3-(2-chloro-6-fluoro-4-trifluoromethylphenoxy)-5-methyl-1H-pyrazole-1-carboxamide (CAS 452100-03-7), 3-[7-fluoro-3-oxo-4-(prop-2-ynyl)-3,4-dihydro-2H-benzo[1,4]oxazin-6-yl]-1,5-dimethyl-6-thioxo-[1,3,5]triazinan-2,4-dione (CAS 451484-50-7), 1,5-dimethyl-6-thioxo-3-(2,2,7-trifluoro-3-oxo-4-(prop-2-ynyl)-3,4-dihydro-2H-benzo[b][1,4]oxazin-6-yl)-1,3,5-triazinane-2,4-dione (CAS 1258836-72-4/trifludimoxazin), 2-(2,2,7-Trifluoro-3-oxo-4-prop-2-ynyl-3,4-dihydro-2H-benzo[1,4]oxazin-6-yl)-4,5,6,7-tetrahydro-isoindole-1,3-dione (CAS 1300118-96-0), 1-Methyl-6-trifluoromethyl-3-(2,2,7-trifluoro-3-oxo-4-prop-2-ynyl-3,4-dihydro-2H-benzo[1,4]oxazin-6-yl)-1H-pyrimidine-2,4-dione, methyl (E)-4-[2-chloro-5-[4-chloro-5-(difluoromethoxy)-1H-methyl-pyrazol-3-yl]-4-fluoro-phenoxy]-3-methoxy-but-2-enoate [CAS 948893-00-3] 3-[7-Chloro-5-fluoro-2-(trifluoromethyl)-1H-benzimidazol-4-yl]-1-methyl-6-(trifluoromethyl)-1H-pyrimidine-2,4-dione (CAS 212754-02-4), and uracils of formula III

-   -   wherein     -   R³⁰ and R³¹ independently of one another are F, Cl or CN;     -   R³² is O or S;     -   R³³ is H, F, Cl, CH₃ or OCH₃;     -   R³⁴ is CH or N;     -   R³⁵ is O or S;     -   R³⁶ is H, CN, CH₃, CF₃, OCH₃, OC₂H₅, SCH₃, SC₂H₅, (CO)OC₂H₅ or         CH₂R³⁸, wherein R³⁸ is F, Cl, OCH₃, SCH₃, SC₂H₅, CH₂F, CH₂Br or         CH₂OH; and     -   R³⁷ is (C₁-C₆-alkyl)amino, (C₁-C₆-dialkyl)amino, (NH)OR³⁹, OH,         OR⁴⁰ or SR⁴⁰         -   wherein R³⁹ is CH₃, C₂H₅ or phenyl; and             -   R⁴⁰ is independently of one another C₁-C₆-alkyl,                 C₂-C₆-alkenyl, C₃-C₆-alkynyl, C₁-C₆-haloalkyl,                 C₁-C₆-alkoxy-C₁-C₆-alkyl,                 C₁-C₆-alkoxy-C₁-C₆-alkoxy-C₁-C₆-alkyl, C₂-C₆-cyanoalkyl,                 C₁-C₄-alkoxy-carbonyl-C₁-C₄-alkyl,                 C₁-C₄-alkyl-carbonyl-amino,                 C₁-C₆-alkylsulfinyl-C₁-C₆-alkyl,                 C₁-C₆-alkyl-sulfonyl-C₁-C₆-alkyl,                 C₁-C₆-dialkoxy-C₁-C₆-alkyl,                 C₁-C₆-alkyl-carbonyloxy-C₁-C₆-alkyl,                 phenyl-carbonyl-C₁-C₆-alkyl,                 tri(C₁-C₃-alkyl)-silyl-C₁-C₆-alkyl,                 tri(C₁-C₃-alkyl)-silyl-C₁-C₆-alkenyl,                 tri(C₁-C₃-alkyl)-silyl-C₁-C₆-alkynyl,                 tri(C₁-C₃-alkyl)-silyl-C₁-C₆-alkoxy-C₁-C₆-alkyl,                 dimethylamino, tetrahydropyranyl,                 tetrahydrofuranyl-C₁-C₃-alkyl,                 phenyl-C₁-C₆-alkoxy-C₁-C₆-alkyl, phenyl-C₁-C₃-alkyl,                 pyridyl-C₁-C₃-alkyl, pyridyl, phenyl,                 -   which pyridyls and phenyls independently of one                     another are substituted by one to five substituents                     selected from the group consisting of halogen,                     C₁-C₃-alkyl or C₁-C₂-haloalkyl;             -   C₃-C₆-cycloalkyl or C₃-C₆-cycloalkyl-C₁-C₄-alkyl,                 -   which cycloalkyls independently of one another are                     unsubstituted or substituted by one to five                     substituents selected from the group consisting of                     halogen, C₁-C₃-alkyl and C₁-C₂-haloalkyl;     -   including their agriculturally acceptable alkali metal salts or         ammonium salts.

Preferred PPO-inhibiting herbicides that can be used according to the present invention are: Acifluorfen, acifluorfen-sodium, azafenidin, bencarbazone, benzfendizone, butafenacil, carfentrazone-ethyl, cinidon-ethyl, flufenpyr-ethyl, flumiclorac-pentyl, flumioxazin, fluoroglycofen-ethyl, fluthiacet-methyl, fomesafen, lactofen, oxadiargyl, oxadiazon, oxyfluorfen, pentoxazone, pyraflufen-ethyl, saflufenacil, sulfentrazone, tiafenacil, ethyl [3-[2-chloro-4-fluoro-5-(1-methyl-6-trifluoromethyl-2,4-dioxo-1,2,3,4-tetrahydropyrimidin-3-yl)phenoxy]-2-pyridyloxy]-acetate (CAS 353292-31-6; S-3100), N-ethyl-3-(2,6-dichloro-4-trifluoromethylphenoxy)-5-methyl-1H-pyrazole-1-carboxamide (CAS 452098-92-9), N-tetrahydrofurfuryl-3-(2,6-dichloro-4-trifluoromethylphenoxy)-5-methyl-1H-pyrazole-1-carboxamide (CAS 915396-43-9), N-ethyl-3-(2-chloro-6-fluoro-4-trifluoromethylphenoxy)-5-methyl-1H-pyrazole-1-carboxamide (CAS 452099-05-7), N-tetrahydrofurfuryl-3-(2-chloro-6-fluoro-4-trifluoromethylphenoxy)-5-methyl-1H-pyrazole-1-carboxamide (CAS 452100-03-7), 3-[7-fluoro-3-oxo-4-(prop-2-ynyl)-3,4-dihydro-2H-benzo[1,4]oxazin-6-yl]-1,5-dimethyl-6-thioxo-[1,3,5]triazinan-2,4-dione (CAS 451484-50-7), 1,5-dimethyl-6-thioxo-3-(2,2,7-trifluoro-3-oxo-4-(prop-2-ynyl)-3,4-dihydro-2H-benzo[b][1,4]oxazin-6-yl)-1,3,5-triazinane-2,4-dione (CAS 1258836-72-4/trifludimoxazin), 2-(2,2,7-Trifluoro-3-oxo-4-prop-2-ynyl-3,4-dihydro-2H-benzo[1,4]oxazin-6-yl)-4,5,6,7-tetrahydro-isoindole-1,3-dione (CAS 1300118-96-0); 1-Methyl-6-trifluoromethyl-3-(2,2,7-trifluoro-3-oxo-4-prop-2-ynyl-3,4-dihydro-2H-benzo[1,4]oxazin-6-yl)-1H-pyrimidine-2,4-dione (CAS 1304113-05-0), 3-[7-Chloro-5-fluoro-2-(trifluoromethyl)-1H-benzimidazol-4-yl]-1-methyl-6-(trifluoromethyl)-1H-pyrimidine-2,4-dione (CAS 212754-02-4)

uracils of formula III.1 (corresponding to uracils of formula III, wherein R³⁰ is F, R³¹ is Cl, R³² is O;

R³³ is H; R³⁴ is CH; R³⁵ is O and R³⁷ is OR⁴⁰)

-   -   wherein     -   R³⁶ is OCH₃, OC₂H₅, SCH₃ or SC₂H₅; and     -   R⁴⁰ is C₁-C₆-alkyl, C₂-C₆-alkenyl, C₃-C₆-alkynyl,         C₁-C₆-haloalkyl, C₁-C₆-alkoxy-C₁-C₆-alkyl,         C₁-C₆-alkoxy-C₁-C₆-alkoxy-C₁-C₆-alkyl, C₁-C₃-cyanoalkyl,         phenyl-C₁-C₃-alkyl, pyridyl-C₁-C₃-alkyl, C₃-C₆-cycloalkyl or         C₃-C₆-cycloalkyl-C₁-C₄-alkyl,         -   which cycloalkyls are unsubstituted or substituted by one to             five substituents selected from the group consisting of             halogen, C₁-C₃-alkyl and C₁-C₂-haloalkyl; and

uracils of formula III.2 (corresponding to uracils of formula III, wherein R³⁰ is F; R³¹ is Cl; R³² is O; R³³ is H; R³⁴ is N; R³⁵ is O and R³⁷ is OR⁴⁰ with R⁴⁰ is C₁-C₆-alkyl)

Particularly preferred PPO-inhibiting herbicides that can be used according to the present invention are:

acifluorfen, acifluorfen-sodium, butafenacil, carfentrazone-ethyl, cinidon-ethyl, flumioxazin, fluthiacet-methyl, fomesafen, lactofen, oxadiargyl, oxyfluorfen, saflufenacil, sulfentrazone, tiafenacil, ethyl [3-[2-chloro-4-fluoro-5-(1-methyl-6-trifluoromethyl-2,4-dioxo-1,2,3,4-tetrahydropyrimidin-3-yl)phenoxy]-2-pyridyloxy]acetate (CAS 353292-31-6; S-3100), 3-[7-fluoro-3-oxo-4-(prop-2-ynyl)-3,4-dihydro-2H-benzo[1,4]oxazin-6-yl]-1,5-dimethyl-6-thioxo-[1,3,5]triazinan-2,4-dione (CAS 451484-50-7), 1,5-dimethyl-6-thioxo-3-(2,2,7-trifluoro-3-oxo-4-(prop-2-ynyl)-3,4-dihydro-2H-benzo[b][1,4]oxazin-6-yl)-1,3,5-triazinane-2,4-dione (CAS 1258836-72-4/trifludimoxazin), and 2-(2,2,7-Trifluoro-3-oxo-4-prop-2-ynyl-3,4-dihydro-2H-benzo[1,4]oxazin-6-yl)-4,5,6,7-tetrahydro-isoindole-1,3-dione (CAS 1300118-96-0), 1-Methyl-6-trifluoromethyl-3-(2,2,7-trifluoro-3-oxo-4-prop-2-ynyl-3,4-dihydro-2H-benzo[1,4]oxazin-6-yl)-1H-pyrimidine-2,4-dione (CAS 1304113-05-0),

uracils of formula II.1.1 (corresponding to uracils of formula III, wherein R³⁰ is F, R³¹ is Cl, R³² is O; R³³ is H; R³⁴ is CH; R³⁵ is O, R³⁶ is OCH₃ and R³⁷ is OR⁴⁰)

-   -   wherein     -   R⁴⁰ is C₁-C₆-alkyl, C₂-C₆-alkenyl, C₃-C₆-alkynyl,         C₁-C₆-haloalkyl, C₁-C₆-alkoxy-C₁-C₆-alkyl,         C₁-C₆-alkoxy-C₁-C₆-alkoxy-C₁-C₆-alkyl, C₁-C₃-cyanoalkyl,         phenyl-C₁-C₃-alkyl, pyridyl-C₁-C₃-alkyl, C₃-C₆-cycloalkyl or         C₃-C₆-cycloalkyl-C₁-C₄-alkyl,         -   which cycloalkyls are unsubstituted or substituted by one to             five substituents         -   selected from the group consisting of halogen, C₁-C₃-alkyl             and C₁-C₂-haloalkyl; is preferably CH₃, CH₂CH₂OC₂H₅,             CH₂CHF₂, cyclohexyl, (1-methylcyclopropyl)methyl or             CH₂(pyridine-4-yl);

uracils of formula III.2.1 (corresponding to uracils of formula III, wherein R³⁰ is F; R³¹ is Cl; R³² is O; R³³ is H; R³⁴ is N; R³⁵ is O and R³⁷ is OR⁴⁰ with R⁴⁰ is CH₃)

and

uracils of formula III.2.2 (corresponding to uracils of formula III, wherein R³⁰ is F; R³¹ is Cl; R³² is O; R³³ is H; R³⁴ is N; R³⁵ is O and R³⁷ is OR⁴⁰ with R⁴⁰ is C₂H₅)

Especially preferred PPO-inhibiting herbicides are the PPO-inhibiting herbicides.1 to A.14 listed below in table A:

TABLE A B.1 acifluorfen B.2 butafenacil B.3 carfentrazone-ethyl B.4 cinidon-ethyl B.5 flumioxazin B.6 fluthiacet-methyl B.7 fomesafen B.8 lactofen B.9 oxadiargyl B.10 oxyfluorfen B.11 saflufenacil B.12 sulfentrazone B.13 ethyl [3-[2-chloro-4-fluoro-5-(1-methyl-6-trifluoromethyl- 2,4-dioxo-1,2,3,4-tetrahydropyrimidin-3-yl)phenoxy]-2- pyridyloxy]acetate (CAS 353292-31-6) B.14 1,5-dimethyl-6-thioxo-3-(2,2,7-trifluoro-3-oxo-4-(prop-2-ynyl)- 3,4-dihydro-2H-benzo[b][1,4]oxazin-6-yl)-1,3,5- triazinane-2,4-dione (CAS 1258836-72-4/trifludimoxazin) B.15 Tiafenacil

Other preferred PPO-inhibiting herbicides include those disclosed in WO2016/120116, the content of which is herein incorporated by reference in its entirety.

Other preferred PPO-inhibiting herbicide include those disclosed in WO2017/202768, the content of which is herein incorporated by reference in its entirety.

More specifically, said PPO-inhibiting herbicide disclosed in W02017/202768 refer to a a uracilpyridine of formula (I)

-   -   wherein the substituents have the following meanings:     -   R¹ hydrogen, NH₂, C₁-C₆-alkyl or C₃-C₆-alkynyl;     -   R² hydrogen, C₁-C₆-alkyl or C₁-C₆-haloalkyl;     -   R³ hydrogen or C₁-C₆-alkyl;     -   R⁴ H or halogen;     -   R⁵ halogen, CN, NO₂, NH₂, CF₃ or C(═S)NH₂;     -   R⁶ H, halogen, CN, C₁-C₃-alkyl, C₁-C₃-haloalkyl, C₁-C₃-alkoxy,         C₁-C₃-haloalkoxy, C₁-C₃-alkylthio, (C₁-C₃-alkyl)amino,         di(C₁-C₃-alkyl)amino, C₁-C₃-alkoxy-C₁-C₃-alkyl,         C₁-C₃-alkoxycarbonyl;     -   R⁷ H, halogen, C₁-C₃-alkyl, C₁-C₃-alkoxy;     -   R⁸ OR⁹, SR⁹, NR¹⁰R¹¹, NR⁹OR⁹, NR⁹S(O)₂R¹⁰ or NR⁹S(O)₂NR¹⁰R¹¹,         wherein         -   R⁹ is hydrogen, C₁-C₆-alkyl, C₃-C₆-alkenyl, C₃-C₆-alkynyl,             C₁-C₆-haloalkyl, C₃-C₆-haloalkenyl, C₃-C₆-haloalkynyl,             C₁-C₆-cyanoalkyl, C₁-C₆-alkoxy-C₁-C₆-alkyl,             C₁-C₆-alkoxy-C₁-C₆-alkoxy-C₁-C₆-alkyl,             di(C₁-C₆-alkoxy)C₁-C₆-alkyl, C₁-C₆-haloalkoxy-C₁-C₆-alkyl,             C₃-C₆-alkenyloxy-C₁-C₆-alkyl,             C₃-C₆-haloalkenyloxy-C₁-C₆-alkyl,             C₃-C₆-alkenyloxy-C₁-C₆-alkoxy-C₁-C₆-alkyl,             C₁-C₆-alkylthio-C₁-C₆-alkyl,             C₁-C₆-alkylsulfinyl-C₁-C₆-alkyl,             C₁-C₆-alkylsulfonyl-C₁-C₆-alkyl,             C₁-C₆-alkylcarbonyl-C₁-C₆-alkyl,             C₁-C₆-alkoxycarbonyl-C₁-C₆-alkyl,             C₁-C₆-haloalkoxycarbonyl-C₁-C₆-alkyl,             C₃-C₆-alkenyloxycarbonyl-C₁-C₆-alkyl,             C₃-C₆-alkynyloxycarbonyl-C₁-C₆-alkyl, amino,             (C₁-C₆-alkyl)amino, di(C₁-C₆-alkyl)amino,             (C₁-C₆-alkylcarbonyl)amino, amino-C₁-C₆-alkyl,             (C₁-C₆-alkyl)amino-C₁-C₆-alkyl,             di(C₁-C₆-alkyl)amino-C₁-C₆-alkyl, aminocarbonyl-C₁-C₆-alkyl,             (C₁-C₆-alkyl)aminocarbonyl-C₁-C₆-alkyl,             di(C₁-C₆-alkyl)aminocarbonyl-C₁-C₆-alkyl,             -   —N═CR¹²R¹³, wherein R¹² and R¹³ independently of one                 another are H, C₁-C₄-alkyl or phenyl;             -   C₃-C₆-cycloalkyl, C₃-C₆-cycloalkyl-C₁-C₆-alkyl,                 C₃-C₆-heterocyclyl, C₃-C₆-heterocyclyl-C₁-C₆-alkyl,                 phenyl, phenyl-C₁-C₄-alkyl or a 5- or 6 membered                 heteroaryl,                 -   wherein each cycloalkyl, heterocyclyl, phenyl or                     heteroaryl ring can be substituted by one to four                     substituents selected from R¹⁴ or a 3- to 7-membered                     carbocyclus,                 -    which carbocyclus optionally has in addition to                     carbon atoms one or two ring members selected from                     the group consisting of —N(R¹²)—, —N═N—, —C(═O)—,                     —O— and —S—, and                 -    which carbocyclus is optionally substituted with                     one to four substituents selected from R¹⁴;                 -     wherein R¹⁴ is halogen, NO₂, CN, C₁-C₄-alkyl,                     C₁-C₄-halo-alkyl, C₁-C₄-alkoxy or                     C₁-C₄-alkoxycarbonyl;         -   R¹⁰, R¹¹ independently of one another are R⁹, or together             form a 3- to 7-membered carbocyclus,             -   which carbocyclus optionally has in addition to carbon                 atoms one or two ring members selected from the group                 consisting of —N(R¹²)—, —N═N—, —C(═O)—, —O— and —S—, and             -   which carbocyclus is optionally substituted with one to                 four substituents selected from R¹⁴;     -   n 1 to 3;     -   Q CH₂, O, S, SO, SO₂, NH or (C₁-C₃-alkyl)N;     -   W O or S;     -   X NH, NCH₃, O or S;     -   Y O or S;     -   Z phenyl, pyridyl, pyridazinyl, pyrimidinyl or pyrazinyl,         -   each of which is optionally substituted by 1 to 4             substituents selected from the group consisting of halogen,             CN, C₁-C₆-alkyl, C₁-C₆-haloalkyl, C₁-C₆-alkoxy,             C₁-C₆-haloalkoxy;     -   including their agriculturally acceptable salts or derivatives,         provided the compounds of formula (I) have a carboxyl group.

The PPO-inhibiting herbicides described above that are useful to carry out the present invention are often best applied in conjunction with one or more other herbicides to obtain control of a wider variety of undesirable vegetation. For example, PPO-inhibiting herbicides may further be used in conjunction with additional herbicides to which the crop plant is naturally tolerant, or to which it is resistant via expression of one or more additional transgenes as mentioned supra, or to which it is resistant via mutagenesis and breeding methods as described. When used in conjunction with other targeting herbicides, the PPO-inhibiting herbicides, to which the plant of the present invention had been made resistant or tolerant, can be formulated with the other herbicide or herbicides, tank mixed with the other herbicide or herbicides, or applied sequentially with the other herbicide or herbicides.

Suitable components for mixtures are, for example, selected from the herbicides of class b1) to b15)

B) herbicides of class b1) to b15):

-   -   b1) lipid biosynthesis inhibitors;     -   b2) acetolactate synthase inhibitors (ALS inhibitors);     -   b3) photosynthesis inhibitors;     -   b4) protoporphyrinogen-IX oxidase inhibitors,     -   b5) bleacher herbicides;     -   b6) enolpyruvyl shikimate 3-phosphate synthase inhibitors (EPSP         inhibitors);     -   b7) glutamine synthetase inhibitors;     -   b8) 7,8-dihydropteroate synthase inhibitors (DHP inhibitors);     -   b9) mitosis inhibitors;     -   b10) inhibitors of the synthesis of very long chain fatty acids         (VLCFA inhibitors);     -   b11) cellulose biosynthesis inhibitors;     -   b12) decoupler herbicides;     -   b13) auxinic herbicides;     -   b14) auxin transport inhibitors; and     -   b15) other herbicides selected from the group consisting of         bromobutide, chlorflurenol, chlorflurenol-methyl, cinmethylin,         cumyluron, dalapon, dazomet, difenzoquat,         difenzoquat-metilsulfate, dimethipin, DSMA, dymron, endothal and         its salts, etobenzanid, flamprop, flamprop-isopropyl,         flamprop-methyl, flamprop-M-isopropyl, flamprop-M-methyl,         flurenol, flurenol-butyl, flurprimidol, fosamine,         fosamine-ammonium, indanofan, indaziflam, maleic hydrazide,         mefluidide, metam, methiozolin (CAS 403640-27-7), methyl azide,         methyl bromide, methyl-dymron, methyl iodide, MSMA, oleic acid,         oxaziclomefone, pelargonic acid, pyributicarb, quinoclamine,         triaziflam, tridiphane and         6-chloro-3-(2-cyclopropyl-6-methylphenoxy-4-pyridazinol (CAS         499223-49-3) and its salts and esters;

including their agriculturally acceptable salts or derivatives.

Examples of herbicides B which can be used in combination with the PPO-inhibiting herbicides according to the present invention are:

b1) from the group of the lipid biosynthesis inhibitors:

ACC-herbicides such as alloxydim, alloxydim-sodium, butroxydim, clethodim, clodinafop, clodinafop-propargyl, cycloxydim, cyhalofop, cyhalofop-butyl, diclofop, diclofop-methyl, fenoxaprop, fenoxaprop-ethyl, fenoxaprop-P, fenoxaprop-P-ethyl, fluazifop, fluazifop-butyl, fluazifop-P, fluazifop-P-butyl, haloxyfop, haloxyfop-methyl, haloxyfop-P, haloxyfop-P-methyl, metamifop, pinoxaden, profoxydim, propaquizafop, quizalofop, quizalofop-ethyl, quizalofop-tefuryl, quizalofop-P, quizalofop-P-ethyl, quizalofop-P-tefuryl, sethoxydim, tepraloxydim, tralkoxydim,

4-(4′-Chloro-4-cyclopropyl-2′-fluoro[1,1′-biphenyl]-3-yl)-5-hydroxy-2,2,6,6-tetramethyl-2H-pyran-3(6H)-one (CAS 1312337-72-6); 4-(2′,4′-Dichloro-4-cyclopropyl[1,1′-biphenyl]-3-yl)-5-hydroxy-2,2,6,6-tetramethyl-2H-pyran-3(6H)-one (CAS 1312337-45-3); 4-(4′-Chloro-4-ethyl-2′-fluoro[1,1′-biphenyl]-3-yl)-5-hydroxy-2,2,6,6-tetramethyl-2H-pyran-3(6H)-one (CAS 1033757-93-5); 4-(2′,4′-Dichloro-4-ethyl[1,1-biphenyl]-3-yl)-2,2,6,6-tetramethyl-2H-pyran-3,5(4H,6H)-dione (CAS 1312340-84-3); 5-(Acetyloxy)-4-(4′-chloro-4-cyclopropyl-2′-fluoro[1,1′-biphenyl]-3-yl)-3,6-dihydro-2,2,6,6-tetramethyl-2H-pyran-3-one (CAS 1312337-48-6); 5-(Acetyloxy)-4-(2′,4′-dichloro-4-cyclopropyl-[1,1-biphenyl]-3-yl)-3,6-dihydro-2,2,6,6-tetramethyl-2H-pyran-3-one; 5-(Acetyloxy)-4-(4′-chloro-4-ethyl-2′-fluoro[1,1′-biphenyl]-3-yl)-3,6-dihydro-2,2,6,6-tetramethyl-2H-pyran-3-one (CAS 1312340-82-1); 5-(Acetyloxy)-4-(2′,4′-dichloro-4-ethyl[1,1′-biphenyl]-3-yl)-3,6-dihydro-2,2,6,6-tetramethyl-2H-pyran-3-one (CAS 1033760-55-2); 4-(4′-Chloro-4-cyclopropyl-2′-fluoro[1,1′-biphenyl]-3-yl)-5,6-dihydro-2,2,6,6-tetramethyl-5-oxo-2H-pyran-3-yl carbonic acid methyl ester (CAS 1312337-51-1); 4-(2′,4′-Dichloro-4-cyclopropyl-[1,1-biphenyl]-3-yl)-5,6-dihydro-2,2,6,6-tetramethyl-5-oxo-2H-pyran-3-yl carbonic acid methyl ester; 4-(4′-Chloro-4-ethyl-2′-fluoro[1,1′-biphenyl]-3-yl)-5,6-dihydro-2,2,6,6-tetramethyl-5-oxo-2H-pyran-3-yl carbonic acid methyl ester (CAS 1312340-83-2); 4-(2′,4′-Dichloro-4-ethyl[1,1′-biphenyl]-3-yl)-5,6-dihydro-2,2,6,6-tetramethyl-5-oxo-2H-pyran-3-yl carbonic acid methyl ester (CAS 1033760-58-5); and non ACC herbicides such as benfuresate, butylate, cycloate, dalapon, dimepiperate, EPTC, esprocarb, ethofumesate, flupropanate, molinate, orbencarb, pebulate, prosulfocarb, TCA, thiobencarb, tiocarbazil, triallate and vernolate;

b2) from the group of the ALS inhibitors:

sulfonylureas such as amidosulfuron, azimsulfuron, bensulfuron, bensulfuron-methyl, chlorimuron, chlorimuron-ethyl, chlorsulfuron, cinosulfuron, cyclosulfamuron, ethametsulfuron, ethametsulfuron-methyl, ethoxysulfuron, flazasulfuron, flucetosulfuron, flupyrsulfuron, flupyrsulfuron-methyl-sodium, foramsulfuron, halosulfuron, halosulfuron-methyl, imazosulfuron, iodosulfuron, iodosulfuron-methyl-sodium, iofensulfuron, iofensulfuron-sodium, mesosulfuron, metazosulfuron, metsulfuron, metsulfuron-methyl, nicosulfuron, orthosulfamuron, oxasulfuron, primisulfuron, primisulfuron-methyl, propyrisulfuron, prosulfuron, pyrazosulfuron, pyrazosulfuron-ethyl, rimsulfuron, sulfometuron, sulfometuron-methyl, sulfosulfuron, thifensulfuron, thifensulfuron-methyl, triasulfuron, tribenuron, tribenuron-methyl, trifloxysulfuron, triflusulfuron, triflusulfuron-methyl and tritosulfuron, imidazolinones such as imazamethabenz, imazamethabenz-methyl, imazamox, imazapic, imazapyr, imazaquin and imazethapyr, triazolopyrimidine herbicides and sulfonanilides such as cloransulam, cloransulam-methyl, diclosulam, flumetsulam, florasulam, metosulam, penoxsulam, pyrimisulfan and pyroxsulam, pyrimidinylbenzoates such as bispyribac, bispyribac-sodium, pyribenzoxim, pyriftalid, pyriminobac, pyriminobac-methyl, pyrithiobac, pyrithiobac-sodium, 4-[[[2-[(4,6-dimethoxy-2-pyrimidinyl)oxy]phenyl]methyl]amino]-benzoic acid-1-methylethyl ester (CAS 420138-41-6), 4-[[[2-[(4,6-dimethoxy-2-pyrimidinyl)oxy]phenyl]methyl]amino]-benzoic acid propyl ester (CAS 420138-40-5), N-(4-bromophenyl)-2-[(4,6-dimethoxy-2-pyrimidinyl)oxy]benzenemethanamine (CAS 420138-01-8), sulfonylaminocarbonyl-triazolinone herbicides such as flucarbazone, flucarbazone-sodium, propoxycarbazone, propoxycarbazone-sodium, thiencarbazone and thiencarbazone-methyl; and triafamone; among these, a preferred embodiment of the invention relates to those compositions comprising at least one imidazolinone herbicide;

b3) from the group of the photosynthesis inhibitors:

amicarbazone, inhibitors of the photosystem II, e.g. triazine herbicides, including of chlorotriazine, triazinones, triazindiones, methylthiotriazines and pyridazinones such as ametryn, atrazine, chloridazone, cyanazine, desmetryn, dimethametryn,hexazinone, metribuzin, prometon, prometryn, propazine, simazine, simetryn, terbumeton, terbuthylazin, terbutryn and trietazin, aryl urea such as chlorobromuron, chlorotoluron, chloroxuron, dimefuron, diuron, fluometuron, isoproturon, isouron, linuron, metamitron, methabenzthiazuron, metobenzuron, metoxuron, monolinuron, neburon, siduron, tebuthiuron and thiadiazuron, phenyl carbamates such as desmedipham, karbutilat, phenmedipham, phenmedipham-ethyl, nitrile herbicides such as bromofenoxim, bromoxynil and its salts and esters, ioxynil and its salts and esters, uraciles such as bromacil, lenacil and terbacil, and bentazon and bentazon-sodium, pyridate, pyridafol, pentanochlor and propanil and inhibitors of the photosystem I such as diquat, diquat-dibromide, paraquat, paraquat-dichloride and paraquat-dimetilsulfate. Among these, a preferred embodiment of the invention relates to those compositions comprising at least one aryl urea herbicide. Among these, likewise a preferred embodiment of the invention relates to those compositions comprising at least one triazine herbicide. Among these, likewise a preferred embodiment of the invention relates to those compositions comprising at least one nitrile herbicide;

b4) from the group of the protoporphyrinogen-IX oxidase inhibitors:

acifluorfen, acifluorfen-sodium, azafenidin, bencarbazone, benzfendizone, bifenox, butafenacil, carfentrazone, carfentrazone-ethyl, chlomethoxyfen, cinidon-ethyl, fluazolate, flufenpyr, flufenpyr-ethyl, flumiclorac, flumiclorac-pentyl, flumioxazin, fluoroglycofen, fluoroglycofen-ethyl, fluthiacet, fluthiacet-methyl, fomesafen, halosafen, lactofen, oxadiargyl, oxadiazon, oxyfluorfen, pentoxazone, profluazol, pyraclonil, pyraflufen, pyraflufen-ethyl, saflufenacil, sulfentrazone, thidiazimin, tiafenacil, ethyl [3-[2-chloro-4-fluoro-5-(1-methyl-6-trifluoromethyl-2,4-dioxo-1,2,3,4-tetrahydropyrimidin-3-yl)phenoxy]-2-pyridyloxy]acetate (CAS 353292-31-6; S-3100, N-ethyl-3-(2,6-dichloro-4-trifluoromethylphenoxy)-5-methyl-1H-pyrazole-1-carboxamide (CAS 452098-92-9), N-tetrahydrofurfuryl-3-(2,6-dichloro-4-trifluoromethylphenoxy)-5-methyl-1H-pyrazole-1-carboxamide (CAS 915396-43-9), N-ethyl-3-(2-chloro-6-fluoro-4-trifluoromethyl-phenoxy)-5-methyl-1H-pyrazole-1-carboxamide (CAS 452099-05-7), N-tetrahydrofurfuryl-3-(2-chloro-6-fluoro-4-trifluoromethylphenoxy)-5-methyl-1H-pyrazole-1-carboxamide (CAS 452100-03-7), 3-[7-fluoro-3-oxo-4-(prop-2-ynyl)-3,4-dihydro-2H-benzo[1,4]oxazin-6-yl]-1,5-dimethyl-6-thioxo-[1,3,5]triazinan-2,4-dione, 1,5-dimethyl-6-thioxo-3-(2,2,7-trifluoro-3-oxo-4-(prop-2-ynyl)-3,4-dihydro-2H-benzo[b][1,4]oxazin-6-yl)-1,3,5-triazinane-2,4-dione (CAS 1258836-72-4/trifludimoxazin), 2-(2,2,7-Trifluoro-3-oxo-4-prop-2-ynyl-3,4-dihydro-2H-benzo[1,4]oxazin-6-yl)-4,5,6,7-tetrahydro-isoindole-1,3-dione, 1-Methyl-6-trifluoromethyl-3-(2,2,7-trifluoro-3-oxo-4-prop-2-ynyl-3,4-dihydro-2H-benzo[1,4]oxazin-6-yl)-1H-pyrimidine-2,4-dione (CAS 1304113-05-0), methyl (E)-4-[2-chloro-5-[4-chloro-5-(difluoromethoxy)-1H-methyl-pyrazol-3-yl]-4-fluoro-phenoxy]-3-methoxy-but-2-enoate [CAS 948893-00-3], and 3-[7-Chloro-5-fluoro-2-(trifluoromethyl)-1H-benzimidazol-4-yl]-1-methyl-6-(trifluoromethyl)-1H-pyrimidine-2,4-dione (CAS 212754-02-4);

b5) from the group of the bleacher herbicides:

PDS inhibitors: beflubutamid, diflufenican, fluridone, flurochloridone, flurtamone, norflurazon, picolinafen, and 4-(3-trifluoromethylphenoxy)-2-(4-trifluoromethylphenyl)pyrimidine (CAS 180608-33-7), HPPD inhibitors: benzobicyclon, benzofenap, clomazone, isoxaflutole, mesotrione, pyrasulfotole, pyrazolynate, pyrazoxyfen, sulcotrione, tefuryltrione, tembotrione, topramezone and bicyclopyrone, bleacher, unknown target: aclonifen, amitrole and flumeturon;

b6) from the group of the EPSP synthase inhibitors:

glyphosate, glyphosate-isopropylammonium, glyposate-potassium and glyphosate-trimesium (sulfosate);

b7) from the group of the glutamine synthase inhibitors:

bilanaphos (bialaphos), bilanaphos-sodium, glufosinate, glufosinate-P and glufosinate-ammonium;

b8) from the group of the DHP synthase inhibitors:

asulam;

b9) from the group of the mitosis inhibitors:

compounds of group K1: dinitroanilines such as benfluralin, butralin, dinitramine, ethalfluralin, fluchloralin, oryzalin, pendimethalin, prodiamine and trifluralin, phosphoramidates such as amiprophos, amiprophos-methyl, and butamiphos, benzoic acid herbicides such as chlorthal, chlorthal-dimethyl, pyridines such as dithiopyr and thiazopyr, benzamides such as propyzamide and tebutam; compounds of group K2: chlorpropham, propham and carbetamide, among these, compounds of group K1, in particular dinitroanilines are preferred;

b10) from the group of the VLCFA inhibitors:

chloroacetamides such as acetochlor, alachlor, butachlor, dimethachlor, dimethenamid, dimethenamid-P, metazachlor, metolachlor, metolachlor-S, pethoxamid, pretilachlor, propachlor, propisochlor and thenylchlor, oxyacetanilides such as flufenacet and mefenacet, acetanilides such as diphenamid, naproanilide and napropamide, tetrazolinones such fentrazamide, and other herbicides such as anilofos, cafenstrole, fenoxasulfone, ipfencarbazone, piperophos, pyroxasulfone and isoxazoline compounds of the formulae II.1, II.2, II.3, II.4, II.5, II.6, II.7, II.8 and II.9

the isoxazoline compounds of the formula (I)I are known in the art, e.g. from WO 2006/024820, WO 2006/037945, WO 2007/071900 and WO 2007/096576;

among the VLCFA inhibitors, preference is given to chloroacetamides and oxyacetamides;

b11) from the group of the cellulose biosynthesis inhibitors:

chlorthiamid, dichlobenil, flupoxam, indaziflam, triaziflam, isoxaben and 1-Cyclohexyl-5-pentafluorphenyloxy-1⁴-[1,2,4,6]thiatriazin-3-ylamine;

b12) from the group of the decoupler herbicides:

dinoseb, dinoterb and DNOC and its salts;

b13) from the group of the auxinic herbicides:

2,4-D and its salts and esters such as clacyfos, 2,4-DB and its salts and esters, aminocyclopyrachlor and its salts and esters, aminopyralid and its salts such as aminopyralid-tris(2-hydroxypropyl)ammonium and its esters, benazolin, benazolin-ethyl, chloramben and its salts and esters, clomeprop, clopyralid and its salts and esters, dicamba and its salts and esters, dichlorprop and its salts and esters, dichlorprop-P and its salts and esters, fluroxypyr, fluroxypyr-butometyl, fluroxypyr-meptyl, halauxifen and its salts and esters (CAS 943832-60-8); MCPA and its salts and esters, MCPA-thioethyl, MCPB and its salts and esters, mecoprop and its salts and esters, mecoprop-P and its salts and esters, picloram and its salts and esters, quinclorac, quinmerac, TBA (2,3,6) and its salts and esters and triclopyr and its salts and esters;

b14) from the group of the auxin transport inhibitors: diflufenzopyr, diflufenzopyr-sodium, naptalam and naptalam-sodium;

b15) from the group of the other herbicides: bromobutide, chlorflurenol, chlorflurenol-methyl, cinmethylin, cumyluron, cyclopyrimorate (CAS 499223-49-3) and its salts and esters, dalapon, dazomet, difenzoquat, difenzoquat-metilsulfate, dimethipin, DSMA, dymron, endothal and its salts, etobenzanid, flamprop, flamprop-isopropyl, flamprop-methyl, flamprop-M-isopropyl, flamprop-M-methyl, flurenol, flurenol-butyl, flurprimidol, fosamine, fosamine-ammonium, indanofan, indaziflam, maleic hydrazide, mefluidide, metam, methiozolin (CAS 403640-27-7), methyl azide, methyl bromide, methyl-dymron, methyl iodide, MSMA, oleic acid, oxaziclomefone, pelargonic acid, pyributicarb, quinoclamine, triaziflam and tridiphane.

Preferred herbicides B that can be used in combination with the PPO-inhibiting herbicides according to the present invention are:

b1) from the group of the lipid biosynthesis inhibitors:

clethodim, clodinafop-propargyl, cycloxydim, cyhalofop-butyl, diclofop-methyl, fenoxaprop-P-ethyl, fluazifop-P-butyl, haloxyfop-P-methyl, metamifop, pinoxaden, profoxydim, propaquizafop, quizalofop-P-ethyl, quizalofop-P-tefuryl, sethoxydim, tepraloxydim, tralkoxydim, 4-(4′-Chloro-4-cyclopropyl-2′-fluoro[1,1′-biphenyl]-3-yl)-5-hydroxy-2,2,6,6-tetramethyl-2H-pyran-3(6H)-one (CAS 1312337-72-6); 4-(2′,4′-Dichloro-4-cyclopropyl[1,1′-biphenyl]-3-yl)-5-hydroxy-2,2,6,6-tetramethyl-2H-pyran-3(6H)-one (CAS 1312337-45-3); 4-(4′-Chloro-4-ethyl-2′-fluoro[1,1′-biphenyl]-3-yl)-5-hydroxy-2,2,6,6-tetramethyl-2H-pyran-3(6H)-one (CAS 1033757-93-5); 4-(2′,4′-Dichloro-4-ethyl[1,1′-biphenyl]-3-yl)-2,2,6,6-tetramethyl-2H-pyran-3,5(4H,6H)-dione (CAS 1312340-84-3); 5-(Acetyloxy)-4-(4′-chloro-4-cyclopropyl-2′-fluoro[1,1′-biphenyl]-3-yl)-3,6-dihydro-2,2,6,6-tetramethyl-2H-pyran-3-one (CAS 1312337-48-6); 5-(Acetyloxy)-4-(2′,4′-dichloro-4-cyclopropyl-[1,1-biphenyl]-3-yl)-3,6-dihydro-2,2,6,6-tetramethyl-2H-pyran-3-one; 5-(Acetyloxy)-4-(4′-chloro-4-ethyl-2′-fluoro[1,1′-biphenyl]-3-yl)-3,6-dihydro-2,2,6,6-tetramethyl-2H-pyran-3-one (CAS 1312340-82-1); 5-(Acetyloxy)-4-(2′,4′-dichloro-4-ethyl[1,1′-biphenyl]-3-yl)-3,6-dihydro-2,2,6,6-tetramethyl-2H-pyran-3-one (CAS 1033760-55-2); 4-(4′-Chloro-4-cyclopropyl-2′-fluoro[1,1′-biphenyl]-3-yl)-5,6-dihydro-2,2,6,6-tetramethyl-5-oxo-2H-pyran-3-yl carbonic acid methyl ester (CAS 1312337-51-1); 4-(2′,4′-Dichloro-4-cyclopropyl-[1,1′-biphenyl]-3-yl)-5,6-dihydro-2,2,6,6-tetramethyl-5-oxo-2H-pyran-3-yl carbonic acid methyl ester; 4-(4′-Chloro-4-ethyl-2′-fluoro[1,1′-biphenyl]-3-yl)-5,6-dihydro-2,2,6,6-tetramethyl-5-oxo-2H-pyran-3-yl carbonic acid methyl ester (CAS 1312340-83-2); 4-(2′,4′-Dichloro-4-ethyl[1,1′-biphenyl]-3-yl)-5,6-dihydro-2,2,6,6-tetramethyl-5-oxo-2H-pyran-3-yl carbonic acid methyl ester (CAS 1033760-58-5); benfuresate, dimepiperate, EPTC, esprocarb, ethofumesate, molinate, orbencarb, prosulfocarb, thiobencarb and triallate;

b2) from the group of the ALS inhibitors:

amidosulfuron, azimsulfuron, bensulfuron-methyl, bispyribac-sodium, chlorimuron-ethyl, chlorsulfuron, cloransulam-methyl, cyclosulfamuron, diclosulam, ethametsulfuron-methyl, ethoxysulfuron, flazasulfuron, florasulam, flucarbazone-sodium, flucetosulfuron, flumetsulam, flupyrsulfuron-methyl-sodium, foramsulfuron, halosulfuron-methyl, imazamethabenz-methyl, imazamox, imazapic, imazapyr, imazaquin, imazethapyr, imazosulfuron, iodosulfuron, iodosulfuron-methyl-sodium, iofensulfuron, iofensulfuron-sodium, mesosulfuron, metazosulfuron, metosulam, metsulfuron-methyl, nicosulfuron, orthosulfamuron, oxasulfuron, penoxsulam, primisulfuron-methyl, propoxycarbazon-sodium, propyrisulfuron, prosulfuron, pyrazosulfuron-ethyl, pyribenzoxim, pyrimisulfan, pyriftalid, pyriminobac-methyl, pyrithiobac-sodium, pyroxsulam, rimsulfuron, sulfometuron-methyl, sulfosulfuron, thiencarbazone-methyl, thifensulfuron-methyl, triasulfuron, tribenuron-methyl, trifloxysulfuron, triflusulfuron-methyl, tritosulfuron and triafamone;

b3) from the group of the photosynthesis inhibitors:

ametryn, amicarbazone, atrazine, bentazone, bentazone-sodium, bromoxynil and its salts and esters, chloridazone, chlorotoluron, cyanazine, desmedipham, diquat-dibromide, diuron, fluometuron, hexazinone, ioxynil and its salts and esters, isoproturon, lenacil, linuron, metamitron, methabenzthiazuron, metribuzin, paraquat, paraquat-dichloride, phenmedipham, propanil, pyridate, simazine, terbutryn, terbuthylazine and thidiazuron;

b4) from the group of the protoporphyrinogen-IX oxidase inhibitors:

acifluorfen, acifluorfen-sodium, azafenidin, bencarbazone, benzfendizone, butafenacil, carfentrazone-ethyl, cinidon-ethyl, flufenpyr-ethyl, flumiclorac-pentyl, flumioxazin, fluoroglycofen-ethyl, fluthiacet-methyl, fomesafen, lactofen, oxadiargyl, oxadiazon, oxyfluorfen, pentoxazone, pyraflufen-ethyl, saflufenacil, sulfentrazone, ethyl [3-[2-chloro-4-fluoro-5-(1-methyl-6-trifluoromethyl-2,4-dioxo-1,2,3,4-tetrahydropyrimidin-3-yl)phenoxy]-2-pyridyloxy]-acetate (CAS 353292-31-6; S-3100), N-ethyl-3-(2,6-dichloro-4-trifluoromethylphenoxy)-5-methyl-1H-pyrazole-1-carboxamide (CAS 452098-92-9), N-tetrahydrofurfuryl-3-(2,6-dichloro-4-trifluoromethylphenoxy)-5-methyl-1H-pyrazole-1-carboxamide (CAS 915396-43-9), N-ethyl-3-(2-chloro-6-fluoro-4-trifluoromethylphenoxy)-5-methyl-1H-pyrazole-1-carboxamide (CAS 452099-05-7), N-tetrahydrofurfuryl-3-(2-chloro-6-fluoro-4-trifluoromethylphenoxy)-5-methyl-1H-pyrazole-1-carboxamide (CAS 452100-03-7), 3-[7-fluoro-3-oxo-4-(prop-2-ynyl)-3,4-dihydro-2H-benzo[1,4]oxazin-6-yl]-1,5-dimethyl-6-thioxo-[1,3,5]triazinan-2,4-dione, 1,5-dimethyl-6-thioxo-3-(2,2,7-trifluoro-3-oxo-4-(prop-2-ynyl)-3,4-dihydro-2H-benzo[b][1,4]oxazin-6-yl)-1,3,5-triazinane-2,4-dione (CAS 1258836-72-4/trifludimoxazin), 2-(2,2,7-Trifluoro-3-oxo-4-prop-2-ynyl-3,4-dihydro-2H-benzo[1,4]oxazin-6-yl)-4,5,6,7-tetrahydro-isoindole-1,3-dione; 1-Methyl-6-trifluoro-methyl-3-(2,2,7-trifluoro-3-oxo-4-prop-2-ynyl-3,4-dihydro-2H-benzo[1,4]oxazin-6-yl)-1H-pyrimidine-2,4-dione, and 3-[7-Chloro-5-fluoro-2-(trifluoromethyl)-1H-benzimidazol-4-yl]-1-methyl-6-(trifluoromethyl)-1H-pyrimidine-2,4-dione (CAS 212754-02-4);

b5) from the group of the bleacher herbicides:

aclonifen, beflubutamid, benzobicyclon, clomazone, diflufenican, flurochloridone, flurtamone, isoxaflutole, mesotrione, norflurazon, picolinafen, pyrasulfotole, pyrazolynate, sulcotrione, tefuryltrione, tembotrione, topramezone, bicyclopyrone, 4-(3-trifluoromethylphenoxy)-2-(4-trifluoromethylphenyl)pyrimidine (CAS 180608-33-7), amitrole and flumeturon;

b6) from the group of the EPSP synthase inhibitors:

glyphosate, glyphosate-isopropylammonium, glyphosate-potassium and glyphosate-trimesium (su Ifosate);

b7) from the group of the glutamine synthase inhibitors:

glufosinate, glufosinate-P, glufosinate-ammonium;

b8) from the group of the DHP synthase inhibitors: asulam;

b9) from the group of the mitosis inhibitors:

benfluralin, dithiopyr, ethalfluralin, oryzalin, pendimethalin, thiazopyr and trifluralin;

b10) from the group of the VLCFA inhibitors:

acetochlor, alachlor, anilofos, butachlor, cafenstrole, dimethenamid, dimethenamid-P, fentrazamide, flufenacet, mefenacet, metazachlor, metolachlor, S-metolachlor, naproanilide, napropamide, pretilachlor, fenoxasulfone, ipfencarbazone, pyroxasulfone thenylchlor and isoxazoline-compounds of the formulae II.1, II.2, II.3, II.4, II.5, II.6, II.7, II.8 and II.9 as mentioned above;

b11) from the group of the cellulose biosynthesis inhibitors: dichlobenil, flupoxam, isoxaben and 1-Cyclohexyl-5-pentafluorphenyloxy-1⁴-[1,2,4,6]thiatriazin-3-ylamine;

b13) from the group of the auxinic herbicides:

2,4-D and its salts and esters, aminocyclopyrachlor and its salts and esters, aminopyralid and its salts such as aminopyralid-tris(2-hydroxypropyl)ammonium and its esters, clopyralid and its salts and esters, dicamba and its salts and esters, dichlorprop-P and its salts and esters, fluroxypyr-meptyl, halauxifen and its salts and esters (CAS 943832-60-8), MCPA and its salts and esters, MCPB and its salts and esters, mecoprop-P and its salts and esters, picloram and its salts and esters, quinclorac, quinmerac and triclopyr and its salts and esters;

b14) from the group of the auxin transport inhibitors: diflufenzopyr and diflufenzopyr-sodium;

b15) from the group of the other herbicides: bromobutide, cinmethylin, cumyluron, cyclopyrimorate (CAS 499223-49-3) and its salts and esters, dalapon, difenzoquat, difenzoquat-metilsulfate, DSMA, dymron (=daimuron), flamprop, flamprop-isopropyl, flamprop-methyl, flamprop-M-isopropyl, flamprop-M-methyl, indanofan, indaziflam, metam, methylbromide, MSMA, oxaziclomefone, pyributicarb, triaziflam and tridiphane.

Particularly preferred herbicides B that can be used in combination with the PPO-inhibiting herbicides according to the present invention are:

b1) from the group of the lipid biosynthesis inhibitors: clodinafop-propargyl, cycloxydim, cyhalofop-butyl, fenoxaprop-P-ethyl, pinoxaden, profoxydim, tepraloxydim, tralkoxydim, 4-(4′-Chloro-4-cyclopropyl-2′-fluoro[1,1′-biphenyl]-3-yl)-5-hydroxy-2,2,6,6-tetramethyl-2H-pyran-3(6H)-one (CAS 1312337-72-6); 4-(2′,4′-Dichloro-4-cyclopropyl[1,1′-biphenyl]-3-yl)-5-hydroxy-2,2,6,6-tetramethyl-2H-pyran-3(6H)-one (CAS 1312337-45-3); 4-(4′-Chloro-4-ethyl-2′-fluoro[1,1′-biphenyl]-3-yl)-5-hydroxy-2,2,6,6-tetramethyl-2H-pyran-3(6H)-one (CAS 1033757-93-5); 4-(2′,4′-Dichloro-4-ethyl[1,1′-biphenyl]-3-yl)-2,2,6,6-tetramethyl-2H-pyran-3,5(4H,6H)-dione (CAS 1312340-84-3); 5-(Acetyloxy)-4-(4′-chloro-4-cyclopropyl-2′-fluoro[1,1′-biphenyl]-3-yl)-3,6-dihydro-2,2,6,6-tetramethyl-2H-pyran-3-one (CAS 1312337-48-6); 5-(Acetyloxy)-4-(2′,4′-dichloro-4-cyclopropyl-[1,1′-biphenyl]-3-yl)-3,6-dihydro-2,2,6,6-tetramethyl-2H-pyran-3-one; 5-(Acetyloxy)-4-(4′-chloro-4-ethyl-2′-fluoro[1,1′-biphenyl]-3-yl)-3,6-dihydro-2,2,6,6-tetramethyl-2H-pyran-3-one (CAS 1312340-82-1); 5-(Acetyloxy)-4-(2′,4′-dichloro-4-ethyl[1,1′-biphenyl]-3-yl)-3,6-dihydro-2,2,6,6-tetramethyl-2H-pyran-3-one (CAS 1033760-55-2); 4-(4′-Chloro-4-cyclopropyl-2′-fluoro[1,1′-biphenyl]-3-yl)-5,6-dihydro-2,2,6,6-tetramethyl-5-oxo-2H-pyran-3-yl carbonic acid methyl ester (CAS 1312337-51-1); 4-(2′,4′-Dichloro-4-cyclopropyl-[1,1′-biphenyl]-3-yl)-5,6-dihydro-2,2,6,6-tetramethyl-5-oxo-2H-pyran-3-yl carbonic acid methyl ester; 4-(4′-Chloro-4-ethyl-2′-fluoro[1,1′-biphenyl]-3-yl)-5,6-dihydro-2,2,6,6-tetramethyl-5-oxo-2H-pyran-3-yl carbonic acid methyl ester (CAS 1312340-83-2); 4-(2′,4′-Dichloro-4-ethyl[1,1′-biphenyl]-3-yl)-5,6-dihydro-2,2,6,6-tetramethyl-5-oxo-2H-pyran-3-yl carbonic acid methyl ester (CAS 1033760-58-5); esprocarb, prosulfocarb, thiobencarb and triallate;

b2) from the group of the ALS inhibitors: bensulfuron-methyl, bispyribac-sodium, cyclosulfamuron, diclosulam, flumetsulam, flupyrsulfuron-methyl-sodium, foramsulfuron, imazamox, imazapic, imazapyr, imazaquin, imazethapyr, imazosulfuron, iodosulfuron, iodosulfuron-methyl-sodium, iofensulfuron, iofensulfuron-sodium, mesosulfuron, metazosulfuron, nicosulfuron, penoxsulam, propoxycarbazon-sodium, propyrisulfuron, pyrazosulfuron-ethyl, pyroxsulam, rimsulfuron, sulfosulfuron, thiencarbazon-methyl, tritosulfuron and triafamone;

b3) from the group of the photosynthesis inhibitors: ametryn, atrazine, diuron, fluometuron, hexazinone, isoproturon, linuron, metribuzin, paraquat, paraquat-dichloride, propanil, terbutryn and terbuthylazine;

b4) from the group of the protoporphyrinogen-IX oxidase inhibitors: acifluorfen, acifluorfen-sodium, butafenacil, cinidon-ethyl, carfentrazone-ethyl, flumioxazin, fluthiacet-methyl, fomesafen, lactofen, oxadiargyl, oxyfluorfen, saflufenacil, sulfentrazone, ethyl [3-[2-chloro-4-fluoro-5-(1-methyl-6-trifluoromethyl-2,4-dioxo-1,2,3,4-tetrahydropyrimidin-3-yl)phenoxy]-2-pyridyloxy]acetate (CAS 353292-31-6; S-3100), 3-[7-fluoro-3-oxo-4-(prop-2-ynyl)-3,4-dihydro-2H-benzo[1,4]oxazin-6-yl]-1,5-dimethyl-6-thioxo-[1,3,5]triazinan-2,4-dione, 1,5-dimethyl-6-thioxo-3-(2,2,7-trifluoro-3-oxo-4-(prop-2-ynyl)-3,4-dihydro-2H-benzo[b][1,4]oxazin-6-yl)-1,3,5-triazinane-2,4-dione (CAS 1258836-72-4/trifludimoxazin), and 2-(2,2,7-Trifluoro-3-oxo-4-prop-2-ynyl-3,4-dihydro-2H-benzo[1,4]oxazin-6-yl)-4,5,6,7-tetrahydro-isoindole-1,3-dione, and 1-Methyl-6-trifluoromethyl-3-(2,2,7-trifluoro-3-oxo-4-prop-2-ynyl-3,4-dihydro-2H-benzo[1,4]oxazin-6-yl)-1H-pyrimidine-2,4-dione;

b5) from the group of the bleacher herbicides: clomazone, diflufenican, flurochloridone, isoxaflutole, mesotrione, picolinafen, sulcotrione, tefuryltrione, tembotrione, topramezone, bicyclopyrone, amitrole and flumeturon;

b6) from the group of the EPSP synthase inhibitors: glyphosate, glyphosate-isopropylammonium and glyphosate-trimesium (sulfosate);

b7) from the group of the glutamine synthase inhibitors: glufosinate, glufosinate-P and glufosinate-ammonium;

b9) from the group of the mitosis inhibitors: pendimethalin and trifluralin;

b10) from the group of the VLCFA inhibitors: acetochlor, cafenstrole, dimethenamid-P, fentrazamide, flufenacet, mefenacet, metazachlor, metolachlor, S-metolachlor, fenoxasulfone, ipfencarbazone and pyroxasulfone; likewise, preference is given to isoxazoline compounds of the formulae II.1, II.2, II.3, II.4, II.5, II.6, II.7, II.8 and II.9 as mentioned above;

b11) from the group of the cellulose biosynthesis inhibitors: isoxaben;

b13) from the group of the auxinic herbicides: 2,4-D and its salts and esters such as clacyfos, and aminocyclopyrachlor and its salts and esters, aminopyralid and its salts and its esters, clopyralid and its salts and esters, dicamba and its salts and esters, fluroxypyr-meptyl, quinclorac and quinmerac;

b14) from the group of the auxin transport inhibitors: diflufenzopyr and diflufenzopyr-sodium,

b15) from the group of the other herbicides: dymron (=daimuron), indanofan, indaziflam, oxaziclomefone and triaziflam.

Moreover, it may be useful to apply the PPO-inhibiting herbicides, when used in combination with a compound B described SUPRA, in combination with safeners. Safeners are chemical compounds which prevent or reduce damage on useful plants without having a major impact on the herbicidal action of herbicides towards unwanted plants. They can be applied either before sowings (e.g. on seed treatments, shoots or seedlings) or in the pre-emergence application or post-emergence application of the useful plant.

Furthermore, the safeners C, the PPO-inhibiting herbicides and/or the herbicides B can be applied simultaneously or in succession.

Suitable safeners are e.g. (quinolin-8-oxy)acetic acids, 1-phenyl-5-haloalkyl-1H-1,2,4-triazol-3-carboxylic acids, 1-phenyl-4,5-dihydro-5-alkyl-1H-pyrazol-3,5-dicarboxylic acids, 4,5-dihydro-5,5-diaryl-3-isoxazol carboxylic acids, dichloroacetamides, alpha-oximinophenylacetonitriles, acetophenonoximes, 4,6-dihalo-2-phenylpyrimidines, N-[[4-(aminocarbonyl)phenyl]sulfonyl]-2-benzoic amides, 1,8-naphthalic anhydride, 2-halo-4-(haloalkyl)-5-thiazol carboxylic acids, phosphorthiolates and N-alkyl-O-phenylcarbamates and their agriculturally acceptable salts and their agriculturally acceptable derivatives such amides, esters, and thioesters, provided they have an acid group.

Examples of preferred safeners C are benoxacor, cloquintocet, cyometrinil, cyprosulfamide, dichlormid, dicyclonon, dietholate, fenchlorazole, fenclorim, flurazole, fluxofenim, furilazole, isoxadifen, mefenpyr, mephenate, naphthalic anhydride, oxabetrinil, 4-(dichloroacetyl)-1-oxa-4-azaspiro[4.5]decane (MON4660, CAS 71526-07-3) and 2,2,5-trimethyl-3-(dichloroacetyl)-1,3-oxazolidine (R-29148, CAS 52836-31-4).

Especially preferred safeners C are benoxacor, cloquintocet, cyprosulfamide, dichlormid, fenchlorazole, fenclorim, flurazole, fluxofenim, furilazole, isoxadifen, mefenpyr, naphthalic anhydride, oxabetrinil, 4-(dichloroacetyl)-1-oxa-4-azaspiro[4.5]decane (MON4660, CAS 71526-07-3) and 2,2,5-trimethyl-3-(dichloroacetyl)-1,3-oxazolidine (R-29148, CAS 52836-31-4).

Particularly preferred safeners C are benoxacor, cloquintocet, cyprosulfamide, dichlormid, fenchlorazole, fenclorim, furilazole, isoxadifen, mefenpyr, naphtalic anhydride, 4-(dichloroacetyl)-1-oxa-4-azaspiro[4.5]decane (MON4660, CAS 71526-07-3), and 2,2,5-trimethyl-3-(dichloroacetyl)-1,3-oxazolidine (R-29148, CAS 52836-31-4).

Also preferred safeners C are benoxacor, cloquintocet, cyprosulfamide, dichlormid, fenchlorazole, fenclorim, furilazole, isoxadifen, mefenpyr, 4-(dichloroacetyl)-1-oxa-4-azaspiro[4.5]decane (MON4660, CAS 71526-07-3) and 2,2,5-trimethyl-3-(dichloroacetyl)-1,3-oxazolidine (R-29148, CAS 52836-31-4).

Particularly preferred safeners C, which, as component C, are constituent of the composition according to the invention are the safeners C as defined above; in particular the safeners C.1-C.12 listed below in table C:

TABLE C Safener C C.1 benoxacor C.2 cloquintocet C.3 cyprosulfamide C.4 dichlormid C.5 fenchlorazole C.6 fenclorim C.7 furilazole C.8 isoxadifen C.9 mefenpyr C.10 naphtalic acid anhydride C.11 4-(dichloroacetyl)-1-oxa-4-azaspiro[4.5]decane (MON4660, CAS 71526-07-3) C.12 2,2,5-trimethyl-3-(dichloro-acetyl)-1,3-oxazolidine (R-29148, CAS 52836-31-4)

The PPO-inhibiting herbicides (compounds A) and the active compounds B of groups b1) to b15) and the active compounds C are known herbicides and safeners, see, for example, The Compendium of Pesticide Common Names (http://www.alanwood.net/pesticides/); Farm Chemicals Handbook 2000 volume 86, Meister Publishing Company, 2000; B. Hock, C. Fedtke, R. R. Schmidt, Herbizide [Herbicides], Georg Thieme Verlag, Stuttgart 1995; W. H. Ahrens, Herbicide Handbook, 7th edition, Weed Science Society of America, 1994; and K. K. Hatzios, Herbicide Handbook, Supplement for the 7th edition, Weed Science Society of America, 1998. 2,2,5-Trimethyl-3-(dichloroacetyl)-1,3-oxazolidine [CAS No. 52836-31-4] is also referred to as R-29148. 4-(Dichloroacetyl)-1-oxa-4-azaspiro[4.5]decane [CAS No. 71526-07-3] is also referred to as AD-67 and MON 4660.

The assignment of the active compounds to the respective mechanisms of action is based on current knowledge. If several mechanisms of action apply to one active compound, this substance was only assigned to one mechanism of action.

Active compounds B and C having a carboxyl group can be employed in the form of the acid, in the form of an agriculturally suitable salt as mentioned above or else in the form of an agriculturally acceptable derivative in the compositions according to the invention.

In the case of dicamba, suitable salts include those, where the counterion is an agriculturally acceptable cation. For example, suitable salts of dicamba are dicamba-sodium, dicamba-potassium, dicamba-methylammonium, dicamba-dimethylammonium, dicamba-isopropylammonium, dicamba-diglycolamine, dicamba-olamine, dicamba-diolamine, dicamba-trolamine, dicamba-N,N-bis-(3-aminopropyl)methylamine and dicamba-diethylenetriamine. Examples of a suitable ester are dicamba-methyl and dicamba-butotyl. Suitable salts of 2,4-D are 2,4-D-ammonium, 2,4-D-dimethylammonium, 2,4-D-diethylammonium, 2,4-D-diethanolammonium (2,4-D-diolamine), 2,4-D-triethanol-ammonium, 2,4-D-isopropylammonium, 2,4-D-triisopropanolammonium, 2,4-D-heptylammonium, 2,4-D-dodecylammonium, 2,4-D-tetradecylammonium, 2,4-D-triethylammonium, 2,4-D-tris(2-hydroxypropyl)ammonium, 2,4-D-tris(isopropyl)ammonium, 2,4-D-trolamine, 2,4-D-lithium, 2,4-D-sodium. Examples of suitable esters of 2,4-D are 2,4-D-butotyl, 2,4-D-2-butoxypropyl, 2,4-D-3-butoxypropyl, 2,4-D-butyl, 2,4-D-ethyl, 2,4-D-ethylhexyl, 2,4-D-isobutyl, 2,4-D-isooctyl, 2,4-D-isopropyl, 2,4-D-meptyl, 2,4-D-methyl, 2,4-D-octyl, 2,4-D-pentyl, 2,4-D-propyl, 2,4-D-tefuryl and clacyfos.

Suitable salts of 2,4-DB are for example 2,4-DB-sodium, 2,4-DB-potassium and 2,4-DB-dimethylammonium. Suitable esters of 2,4-DB are for example 2,4-DB-butyl and 2,4-DB-isoctyl.

Suitable salts of dichlorprop are for example dichlorprop-sodium, dichlorprop-potassium and dichlorprop-dimethylammonium. Examples of suitable esters of dichlorprop are dichlorprop-butotyl and dichlorprop-isoctyl.

Suitable salts and esters of MCPA include MCPA-butotyl, MCPA-butyl, MCPA-dimethyl-ammonium, MCPA-diolamine, MCPA-ethyl, MCPA-thioethyl, MCPA-2-ethylhexyl, MCPA-isobutyl, MCPA-isoctyl, MCPA-isopropyl, MCPA-isopropylammonium, MCPA-methyl, MCPA-olamine, MCPA-potassium, MCPA-sodium and MCPA-trolamine.

A suitable salt of MCPB is MCPB sodium. A suitable ester of MCPB is MCPB-ethyl. Suitable salts of clopyralid are clopyralid-potassium, clopyralid-olamine and clopyralid-tris-(2-hydroxypropyl)ammonium. Example of suitable esters of clopyralid is clopyralid-methyl. Examples of a suitable ester of fluroxypyr are fluroxypyr-meptyl and fluroxypyr-2-butoxy-1-methylethyl, wherein fluroxpyr-meptyl is preferred.

Suitable salts of picloram are picloram-dimethylammonium, picloram-potassium, picloram-triisopropanolammonium, picloram-triisopropylammonium and picloram-trolamine. A suitable ester of picloram is picloram-isoctyl.

A suitable salt of triclopyr is triclopyr-triethylammonium. Suitable esters of triclopyr are for example triclopyr-ethyl and triclopyr-butotyl.

Suitable salts and esters of chloramben include chloramben-ammonium, chloramben-diolamine, chloramben-methyl, chloramben-methylammonium and chloramben-sodium.

Suitable salts and esters of 2,3,6-TBA include 2,3,6-TBA-dimethylammonium, 2,3,6-TBA-lithium, 2,3,6-TBA-potassium and 2,3,6-TBA-sodium.

Suitable salts and esters of aminopyralid include aminopyralid-potassium and aminopyralid-tris(2-hydroxypropyl)ammonium.

Suitable salts of glyphosate are for example glyphosate-ammonium, glyphosate-diammonium, glyphoste-dimethylammonium, glyphosate-isopropylammonium, glyphosate-potassium, glyphosate-sodium, glyphosate-trimesium as well as the ethanolamine and diethanolamine salts, preferably glyphosate-diammonium, glyphosate-isopropylammonium and glyphosate-trimesium (sulfosate).

A suitable salt of glufosinate is for example glufosinate-ammonium.

A suitable salt of glufosinate-P is for example glufosinate-P-ammonium.

Suitable salts and esters of bromoxynil are for example bromoxynil-butyrate, bromoxynil-heptanoate, bromoxynil-octanoate, bromoxynil-potassium and bromoxynil-sodium.

Suitable salts and esters of ioxonil are for example ioxonil-octanoate, ioxonil-potassium and ioxonil-sodium.

Suitable salts and esters of mecoprop include mecoprop-butotyl, mecoprop-dimethylammonium, mecoprop-diolamine, mecoprop-ethadyl, mecoprop-2-ethylhexyl, mecoprop-isoctyl, mecoprop-methyl, mecoprop-potassium, mecoprop-sodium and mecoprop-trolamine.

Suitable salts of mecoprop-P are for example mecoprop-P-butotyl, mecoprop-P-dimethylammonium, mecoprop-P-2-ethylhexyl, mecoprop-P-isobutyl, mecoprop-P-potassium and mecoprop-P-sodium.

A suitable salt of diflufenzopyr is for example diflufenzopyr-sodium.

A suitable salt of naptalam is for example naptalam-sodium.

Suitable salts and esters of aminocyclopyrachlor are for example aminocyclopyrachlor-dimethylammonium, aminocyclopyrachlor-methyl, aminocyclopyrachlor-triisopropanolammonium, aminocyclopyrachlor-sodium and aminocyclopyrachlor-potassium.

A suitable salt of quinclorac is for example quinclorac-dimethylammonium.

A suitable salt of quinmerac is for example quinclorac-dimethylammonium.

A suitable salt of imazamox is for example imazamox-ammonium.

Suitable salts of imazapic are for example imazapic-ammonium and imazapic-isopropylammonium.

Suitable salts of imazapyr are for example imazapyr-ammonium and imazapyr-isopropylammonium.

A suitable salt of imazaquin is for example imazaquin-ammonium.

Suitable salts of imazethapyr are for example imazethapyr-ammonium and imazethapyr-isopropylammonium.

A suitable salt of topramezone is for example topramezone-sodium.

The preferred embodiments of the invention mentioned herein below have to be understood as being preferred either independently from each other or in combination with one another.

According to a preferred embodiment of the invention, the composition comprises as component B at least one, preferably exactly one herbicide B.

According to another preferred embodiment of the invention, the composition comprises at least two, preferably exactly two, herbicides B different from each other.

According to another preferred embodiment of the invention, the composition comprises at least three, preferably exactly three, herbicides B different from each other.

According to another preferred embodiment of the invention, the composition comprises as component A at least one, preferably exactly one PPO-inhibiting herbicide, preferably acifluorfen, acifluorfen-sodium, butafenacil, cinidon-ethyl, carfentrazone-ethyl, flumioxazin, fluthiacet-methyl, fomesafen, lactofen, oxadiargyl, oxyfluorfen, saflufenacil, sulfentrazone, ethyl [3-[2-chloro-4-fluoro-5-(1-methyl-6-trifluoromethyl-2,4-dioxo-1,2,3,4-tetrahydropyrimidin-3-yl)phenoxy]-2-pyridyloxy]acetate (CAS 353292-31-6; S-3100, 1,5-dimethyl-6-thioxo-3-(2,2,7-trifluoro-3-oxo-4-(prop-2-ynyl)-3,4-dihydro-2H-benzo[b][1,4]oxazin-6-yl)-1,3,5-triazinane-2,4-dione (CAS 1258836-72-4/trifludimoxazin), especially preferred saflufenacil, 1,5-dimethyl-6-thioxo-3-(2,2,7-trifluoro-3-oxo-4-(prop-2-ynyl)-3,4-dihydro-2H-benzo[b][1,4]oxazin-6-yl)-1,3,5-triazinane-2,4-dione (CAS 1258836-72-4/trifludimoxazin), and as component B at least one, preferably exactly one, herbicide B.

According to another preferred embodiment of the invention, the composition comprises as component A at least one, preferably exactly preferably exactly one PPO-inhibiting herbicide, preferably acifluorfen, acifluorfen-sodium, butafenacil, cinidon-ethyl, carfentrazone-ethyl, flumioxazin, fluthiacet-methyl, fomesafen, lactofen, oxadiargyl, oxyfluorfen, saflufenacil, sulfentrazone, ethyl [3-[2-chloro-4-fluoro-5-(1-methyl-6-trifluoromethyl-2,4-dioxo-1,2,3,4-tetrahydropyrimidin-3-yl)phenoxy]-2-pyridyloxy]acetate (CAS 353292-31-6; S-3100), 1,5-dimethyl-6-thioxo-3-(2,2,7-trifluoro-3-oxo-4-(prop-2-ynyl)-3,4-dihydro-2H-benzo[b][1,4]oxazin-6-yl)-1,3,5-triazinane-2,4-dione (CAS 1258836-72-4/trifludimoxazin), especially preferred saflufenacil, 1,5-dimethyl-6-thioxo-3-(2,2,7-trifluoro-3-oxo-4-(prop-2-ynyl)-3,4-dihydro-2H-benzo[b][1,4]oxazin-6-yl)-1,3,5-triazinane-2,4-dione (CAS 1258836-72-4/trifludimoxazin), and at least two, preferably exactly two, herbicides B different from each other.

According to another preferred embodiment of the invention, the composition comprises as component A at least one, preferably exactly preferably exactly one PPO-inhibiting herbicide, preferably acifluorfen, acifluorfen-sodium, butafenacil, cinidon-ethyl, carfentrazone-ethyl, flumioxazin, fluthiacet-methyl, fomesafen, lactofen, oxadiargyl, oxyfluorfen, saflufenacil, sulfentrazone, ethyl [3-[2-chloro-4-fluoro-5-(1-methyl-6-trifluoromethyl-2,4-dioxo-1,2,3,4-tetrahydropyrimidin-3-yl)phenoxy]-2-pyridyloxy]acetate (CAS 353292-31-6; S-3100), 1,5-dimethyl-6-thioxo-3-(2,2,7-trifluoro-3-oxo-4-(prop-2-ynyl)-3,4-dihydro-2H-benzo[b][1,4]oxazin-6-yl)-1,3,5-triazinane-2,4-dione (CAS 1258836-72-4/trifludimoxazin), especially preferred saflufenacil, 1,5-dimethyl-6-thioxo-3-(2,2,7-trifluoro-3-oxo-4-(prop-2-ynyl)-3,4-dihydro-2H-benzo[b][1,4]oxazin-6-yl)-1,3,5-triazinane-2,4-dione (CAS 1258836-72-4/trifludimoxazin) and at least three, preferably exactly three, herbicides B different from each other.

According to another preferred embodiment of the invention, the composition comprises, in addition to a PPO-inhibiting herbicide, preferably acifluorfen, acifluorfen-sodium, butafenacil, cinidon-ethyl, carfentrazone-ethyl, flumioxazin, fluthiacet-methyl, fomesafen, lactofen, oxadiargyl, oxyfluorfen, saflufenacil, sulfentrazone, ethyl [3-[2-chloro-4-fluoro-5-(1-methyl-6-trifluoromethyl-2,4-dioxo-1,2,3,4-tetrahydropyrimidin-3-yl)phenoxy]-2-pyridyloxy]acetate (CAS 353292-31-6; S-3100), 1,5-dimethyl-6-thioxo-3-(2,2,7-trifluoro-3-oxo-4-(prop-2-ynyl)-3,4-dihydro-2H-benzo[b][1,4]oxazin-6-yl)-1,3,5-triazinane-2,4-dione (CAS 1258836-72-4/trifludimoxazin), especially preferred saflufenacil, 1,5-dimethyl-6-thioxo-3-(2,2,7-trifluoro-3-oxo-4-(prop-2-ynyl)-3,4-dihydro-2H-benzo[b][1,4]oxazin-6-yl)-1,3,5-triazinane-2,4-dione (CAS 1258836-72-4/trifludimoxazin), at least one and especially exactly one herbicidally active compound from group b1), in particular selected from the group consisting of clethodim, clodinafop-propargyl, cycloxydim, cyhalofop-butyl, fenoxaprop-P-ethyl, fluazifop, pinoxaden, profoxydim, quizalofop, sethoxydim, tepraloxydim, tralkoxydim, esprocarb, prosulfocarb, thiobencarb and triallate.

According to another preferred embodiment of the invention, the composition comprises, in addition to a PPO-inhibiting herbicide, preferably acifluorfen, acifluorfen-sodium, butafenacil, cinidon-ethyl, carfentrazone-ethyl, flumioxazin, fluthiacet-methyl, fomesafen, lactofen, oxadiargyl, oxyfluorfen, saflufenacil, sulfentrazone, ethyl [3-[2-chloro-4-fluoro-5-(1-methyl-6-trifluoromethyl-2,4-dioxo-1,2,3,4-tetrahydropyrimidin-3-yl)phenoxy]-2-pyridyloxy]acetate (CAS 353292-31-6; S-3100, 1,5-dimethyl-6-thioxo-3-(2,2,7-trifluoro-3-oxo-4-(prop-2-ynyl)-3,4-dihydro-2H-benzo[b][1,4]oxazin-6-yl)-1,3,5-triazinane-2,4-dione (CAS 1258836-72-4/trifludimoxazin) especially preferred saflufenacil, 1,5-dimethyl-6-thioxo-3-(2,2,7-trifluoro-3-oxo-4-(prop-2-ynyl)-3,4-dihydro-2H-benzo[b][1,4]oxazin-6-yl)-1,3,5-triazinane-2,4-dione (CAS 1258836-72-4/trifludimoxazin), at least one and especially exactly one herbicidally active compound from group b2), in particular selected from the group consisting of bensulfuron-methyl, bispyribac-sodium, cloransulam-methyl, cyclosulfamuron, diclosulam, flumetsulam, flupyrsulfuron-methyl-sodium, foramsulfuron, halosulfuron-methyl, imazamox, imazapic, imazapyr, imazaquin, imazethapyr, imazosulfuron, iodosulfuron, iodosulfuron-methyl-sodium, mesosulfuron-methyl, metazosulfuron, nicosulfuron, penoxsulam, propoxycarbazon-sodium, pyrazosulfuron-ethyl, pyrithiobac-sodium, pyroxsulam, rimsulfuron, sulfosulfuron, thiencarbazon-methyl, thifensulfuron-methyl, trifloxysulfuron and tritosulfuron.

According to another preferred embodiment of the invention, the composition comprises, in addition to a a PPO-inhibiting herbicide, preferably acifluorfen, acifluorfen-sodium, butafenacil, cinidon-ethyl, carfentrazone-ethyl, flumioxazin, fluthiacet-methyl, fomesafen, lactofen, oxadiargyl, oxyfluorfen, saflufenacil, sulfentrazone, ethyl [3-[2-chloro-4-fluoro-5-(1-methyl-6-trifluoromethyl-2,4-dioxo-1,2,3,4-tetrahydropyrimidin-3-yl)phenoxy]-2-pyridyloxy]acetate (CAS 353292-31-6; S-3100), 1,5-dimethyl-6-thioxo-3-(2,2,7-trifluoro-3-oxo-4-(prop-2-ynyl)-3,4-dihydro-2H-benzo[b][1,4]oxazin-6-yl)-1,3,5-triazinane-2,4-dione (CAS 1258836-72-4/trifludimoxazin), especially preferred saflufenacil, 1,5-dimethyl-6-thioxo-3-(2,2,7-trifluoro-3-oxo-4-(prop-2-ynyl)-3,4-dihydro-2H-benzo[b][1,4]oxazin-6-yl)-1,3,5-triazinane-2,4-dione (CAS 1258836-72-4/trifludimoxazin), at least one and especially exactly one herbicidally active compound from group b3), in particular selected from the group consisting of ametryn, atrazine, bentazon, bromoxynil, diuron, fluometuron, hexazinone, isoproturon, linuron, metribuzin, paraquat, paraquat-dichloride, prometryne, propanil, terbutryn and terbuthylazine.

According to another preferred embodiment of the invention, the composition comprises, in addition to a a PPO-inhibiting herbicide, preferably acifluorfen, acifluorfen-sodium, butafenacil, cinidon-ethyl, carfentrazone-ethyl, flumioxazin, fluthiacet-methyl, fomesafen, lactofen, oxadiargyl, oxyfluorfen, saflufenacil, sulfentrazone, ethyl [3-[2-chloro-4-fluoro-5-(1-methyl-6-trifluoromethyl-2,4-dioxo-1,2,3,4-tetrahydropyrimidin-3-yl)phenoxy]-2-pyridyloxy]acetate (CAS 353292-31-6; S-3100), 1,5-dimethyl-6-thioxo-3-(2,2,7-trifluoro-3-oxo-4-(prop-2-ynyl)-3,4-dihydro-2H-benzo[b][1,4]oxazin-6-yl)-1,3,5-triazinane-2,4-dione (CAS 1258836-72-4/trifludimoxazin), especially preferred saflufenacil, 1,5-dimethyl-6-thioxo-3-(2,2,7-trifluoro-3-oxo-4-(prop-2-ynyl)-3,4-dihydro-2H-benzo[b][1,4]oxazin-6-yl)-1,3,5-triazinane-2,4-dione (CAS 1258836-72-4/trifludimoxazin), at least one and especially exactly one herbicidally active compound from group b4), in particular selected from the group consisting of acifluorfen, acifluorfen-sodium, azafenidin, bencarbazone, benzfendizone, bifenox, butafenacil, carfentrazone, carfentrazone-ethyl, chlomethoxyfen, cinidon-ethyl, fluazolate, flufenpyr, flufenpyr-ethyl, flumiclorac, flumiclorac-pentyl, flumioxazin, fluoroglycofen, fluoroglycofen-ethyl, fluthiacet, fluthiacet-methyl, fomesafen, halosafen, lactofen, oxadiargyl, oxadiazon, oxyfluorfen, pentoxazone, profluazol, pyraclonil, pyraflufen, pyraflufen-ethyl, saflufenacil, sulfentrazone, thidiazimin, tiafenacil, ethyl [3-[2-chloro-4-fluoro-5-(1-methyl-6-trifluoromethyl-2,4-dioxo-1,2,3,4-tetrahydropyrimidin-3-yl)phenoxy]-2-pyridyloxy]acetate (CAS 353292-31-6; S-3100), N-ethyl-3-(2,6-dichloro-4-trifluoro-methylphenoxy)-5-methyl-1H-pyrazole-1-carboxamide (CAS 452098-92-9), N-tetrahydrofurfuryl-3-(2,6-dichloro-4-trifluoromethylphenoxy)-5-methyl-1H-pyrazole-1-carboxamide (CAS 915396-43-9), N-ethyl-3-(2-chloro-6-fluoro-4-trifluoromethylphenoxy)-5-methyl-1H-pyrazole-1-carboxamide (CAS 452099-05-7), N-tetrahydrofurfuryl-3-(2-chloro-6-fluoro-4-trifluoro-methylphenoxy)-5-methyl-1H-pyrazole-1-carboxamide (CAS 452100-03-7), 3-[7-fluoro-3-oxo-4-(prop-2-ynyl)-3,4-dihydro-2H-benzo[1,4]oxazin-6-yl]-1,5-dimethyl-6-thioxo-[1,3,5]triazinan-2,4-dione, 1,5-dimethyl-6-thioxo-3-(2,2,7-trifluoro-3-oxo-4-(prop-2-ynyl)-3,4-dihydro-2H-benzo[b][1,4]oxazin-6-yl)-1,3,5-triazinane-2,4-dione (CAS 1258836-72-4/trifludimoxazin), 2-(2,2,7-Trifluoro-3-oxo-4-prop-2-ynyl-3,4-dihydro-2H-benzo[1,4]oxazin-6-yl)-4,5,6,7-tetrahydro-isoindole-1,3-dione, 1-Methyl-6-trifluoromethyl-3-(2,2,7-trifluoro-3-oxo-4-prop-2-ynyl-3,4-dihydro-2H-benzo[1,4]oxazin-6-yl)-1H-pyrimidine-2,4-dione, methyl (E)-4-[2-chloro-5-[4-chloro-5-(difluoromethoxy)-1H-methyl-pyrazol-3-yl]-4-fluoro-phenoxy]-3-methoxy-but-2-enoate [CAS 948893-00-3], 3-[7-Chloro-5-fluoro-2-(trifluoromethyl)-1H-benzimidazol-4-yl]-1-methyl-6-(trifluoromethyl)-1H-pyrimidine-2,4-dione (CAS 212754-02-4).

According to another preferred embodiment of the invention, the composition comprises, in addition to a a PPO-inhibiting herbicide, preferably acifluorfen, acifluorfen-sodium, butafenacil, cinidon-ethyl, carfentrazone-ethyl, flumioxazin, fluthiacet-methyl, fomesafen, lactofen, oxadiargyl, oxyfluorfen, saflufenacil, sulfentrazone, ethyl [3-[2-chloro-4-fluoro-5-(1-methyl-6-trifluoromethyl-2,4-dioxo-1,2,3,4-tetrahydropyrimidin-3-yl)phenoxy]-2-pyridyloxy]acetate (CAS 353292-31-6; S-3100), 1,5-dimethyl-6-thioxo-3-(2,2,7-trifluoro-3-oxo-4-(prop-2-ynyl)-3,4-dihydro-2H-benzo[b][1,4]oxazin-6-yl)-1,3,5-triazinane-2,4-dione (CAS 1258836-72-4/trifludimoxazin), especially preferred saflufenacil, 1,5-dimethyl-6-thioxo-3-(2,2,7-trifluoro-3-oxo-4-(prop-2-ynyl)-3,4-dihydro-2H-benzo[b][1,4]oxazin-6-yl)-1,3,5-triazinane-2,4-dione (CAS 1258836-72-4/trifludimoxazin), at least one and especially exactly one herbicidally active compound from group b5), in particular selected from the group consisting of clomazone, diflufenican, flurochloridone, isoxaflutole, mesotrione, picolinafen, sulcotrione, tefuryltrione, tembotrione, topramezone, bicyclopyrone, amitrole and flumeturon.

According to another preferred embodiment of the invention, the composition comprises, in addition to a a PPO-inhibiting herbicide, preferably acifluorfen, acifluorfen-sodium, butafenacil, cinidon-ethyl, carfentrazone-ethyl, flumioxazin, fluthiacet-methyl, fomesafen, lactofen, oxadiargyl, oxyfluorfen, saflufenacil, sulfentrazone, ethyl [3-[2-chloro-4-fluoro-5-(1-methyl-6-trifluoromethyl-2,4-dioxo-1,2,3,4-tetrahydropyrimidin-3-yl)phenoxy]-2-pyridyloxy]acetate (CAS 353292-31-6; 5-3100), 1,5-dimethyl-6-thioxo-3-(2,2,7-trifluoro-3-oxo-4-(prop-2-ynyl)-3,4-dihydro-2H-benzo[b][1,4]oxazin-6-yl)-1,3,5-triazinane-2,4-dione (CAS 1258836-72-4/trifludimoxazin), especially preferred saflufenacil, 1,5-dimethyl-6-thioxo-3-(2,2,7-trifluoro-3-oxo-4-(prop-2-ynyl)-3,4-dihydro-2H-benzo[b][1,4]oxazin-6-yl)-1,3,5-triazinane-2,4-dione (CAS 1258836-72-4/trifludimoxazin), at least one and especially exactly one herbicidally active compound from group b6), in particular selected from the group consisting of glyphosate, glyphosate-isopropylammonium and glyphosate-trimesium (sulfosate).

According to another preferred embodiment of the invention, the composition comprises, in addition to a a PPO-inhibiting herbicide, preferably acifluorfen, acifluorfen-sodium, butafenacil, cinidon-ethyl, carfentrazone-ethyl, flumioxazin, fluthiacet-methyl, fomesafen, lactofen, oxadiargyl, oxyfluorfen, saflufenacil, sulfentrazone, ethyl [3-[2-chloro-4-fluoro-5-(1-methyl-6-trifluoromethyl-2,4-dioxo-1,2,3,4-tetrahydropyrimidin-3-yl)phenoxy]-2-pyridyloxy]acetate (CAS 353292-31-6; S-3100), 1,5-dimethyl-6-thioxo-3-(2,2,7-trifluoro-3-oxo-4-(prop-2-ynyl)-3,4-dihydro-2H-benzo[b][1,4]oxazin-6-yl)-1,3,5-triazinane-2,4-dione (CAS 1258836-72-4/trifludimoxazin), especially preferred saflufenacil, 1,5-dimethyl-6-thioxo-3-(2,2,7-trifluoro-3-oxo-4-(prop-2-ynyl)-3,4-dihydro-2H-benzo[b][1,4]oxazin-6-yl)-1,3,5-triazinane-2,4-dione (CAS 1258836-72-4/trifludimoxazin), at least one and especially exactly one herbicidally active compound from group b7), in particular selected from the group consisting of glufosinate, glufosinate-P and glufosinate-ammonium.

According to another preferred embodiment of the invention, the composition comprises, in addition to a a PPO-inhibiting herbicide, preferably acifluorfen, acifluorfen-sodium, butafenacil, cinidon-ethyl, carfentrazone-ethyl, flumioxazin, fluthiacet-methyl, fomesafen, lactofen, oxadiargyl, oxyfluorfen, saflufenacil, sulfentrazone, ethyl [3-[2-chloro-4-fluoro-5-(1-methyl-6-trifluoromethyl-2,4-dioxo-1,2,3,4-tetrahydropyrimidin-3-yl)phenoxy]-2-pyridyloxy]acetate (CAS 353292-31-6; S-3100, 1,5-dimethyl-6-thioxo-3-(2,2,7-trifluoro-3-oxo-4-(prop-2-ynyl)-3,4-dihydro-2H-benzo[b][1,4]oxazin-6-yl)-1,3,5-triazinane-2,4-dione (CAS 1258836-72-4/trifludimoxazin) especially preferred saflufenacil, 1,5-dimethyl-6-thioxo-3-(2,2,7-trifluoro-3-oxo-4-(prop-2-ynyl)-3,4-dihydro-2H-benzo[b][1,4]oxazin-6-yl)-1,3,5-triazinane-2,4-dione (CAS 1258836-72-4/trifludimoxazin), at least one and especially exactly one herbicidally active compound from group b9), in particular selected from the group consisting of pendimethalin and trifluralin.

According to another preferred embodiment of the invention, the composition comprises, in addition to a PPO-inhibiting herbicide, preferably acifluorfen, acifluorfen-sodium, butafenacil, cinidon-ethyl, carfentrazone-ethyl, flumioxazin, fluthiacet-methyl, fomesafen, lactofen, oxadiargyl, oxyfluorfen, saflufenacil, sulfentrazone, ethyl [3-[2-chloro-4-fluoro-5-(1-methyl-6-trifluoromethyl-2,4-dioxo-1,2,3,4-tetrahydropyrimidin-3-yl)phenoxy]-2-pyridyloxy]acetate (CAS 353292-31-6; S-3100, 1,5-dimethyl-6-thioxo-3-(2,2,7-trifluoro-3-oxo-4-(prop-2-ynyl)-3,4-dihydro-2H-benzo[b][1,4]oxazin-6-yl)-1,3,5-triazinane-2,4-dione (CAS 1258836-72-4/trifludimoxazin)) especially preferred saflufenacil, 1,5-dimethyl-6-thioxo-3-(2,2,7-trifluoro-3-oxo-4-(prop-2-ynyl)-3,4-dihydro-2H-benzo[b][1,4]oxazin-6-yl)-1,3,5-triazinane-2,4-dione (CAS 1258836-72-4/trifludimoxazin), at least one and especially exactly one herbicidally active compound from group b10), in particular selected from the group consisting of acetochlor, cafenstrole, dimethenamid-P, fentrazamide, flufenacet, mefenacet, metazachlor, metolachlor, S-metolachlor, fenoxasulfone and pyroxasulfone. Likewise, preference is given to compositions comprising in addition to a a PPO-inhibiting herbicide, preferably acifluorfen, acifluorfen-sodium, butafenacil, cinidon-ethyl, carfentrazone-ethyl, flumioxazin, fluthiacet-methyl, fomesafen, lactofen, oxadiargyl, oxyfluorfen, saflufenacil, sulfentrazone, ethyl [3-[2-chloro-4-fluoro-5-(1-methyl-6-trifluoromethyl-2,4-dioxo-1,2,3,4-tetrahydropyrimidin-3-yl)phenoxy]-2-pyridyloxy]acetate (CAS 353292-31-6; S-3100), 1,5-dimethyl-6-thioxo-3-(2,2,7-trifluoro-3-oxo-4-(prop-2-ynyl)-3,4-dihydro-2H-benzo[b][1,4]oxazin-6-yl)-1,3,5-triazinane-2,4-dione (CAS 1258836-72-4/trifludimoxazin), especially preferred saflufenacil, 1,5-dimethyl-6-thioxo-3-(2,2,7-trifluoro-3-oxo-4-(prop-2-ynyl)-3,4-dihydro-2H-benzo[b][1,4]oxazin-6-yl)-1,3,5-triazinane-2,4-dione (CAS 1258836-72-4/trifludimoxazin), at least one and especially exactly one herbicidally active compound from group b10), in particular selected from the group consisting of isoxazoline compounds of the formulae II.1, II.2, II.3, II.4, II.5, II.6, II.7, II.8 and II.9, as defined above.

According to another preferred embodiment of the invention, the composition comprises, in addition to a PPO-inhibiting herbicide, preferably acifluorfen, acifluorfen-sodium, butafenacil, cinidon-ethyl, carfentrazone-ethyl, flumioxazin, fluthiacet-methyl, fomesafen, lactofen, oxadiargyl, oxyfluorfen, saflufenacil, sulfentrazone, ethyl [3-[2-chloro-4-fluoro-5-(1-methyl-6-trifluoromethyl-2,4-dioxo-1,2,3,4-tetrahydropyrimidin-3-yl)phenoxy]-2-pyridyloxy]acetate (CAS 353292-31-6; S-3100, 1,5-dimethyl-6-thioxo-3-(2,2,7-trifluoro-3-oxo-4-(prop-2-ynyl)-3,4-dihydro-2H-benzo[b][1,4]oxazin-6-yl)-1,3,5-triazinane-2,4-dione (CAS 1258836-72-4/trifludimoxazin), especially preferred saflufenacil, 1,5-dimethyl-6-thioxo-3-(2,2,7-trifluoro-3-oxo-4-(prop-2-ynyl)-3,4-dihydro-2H-benzo[b][1,4]oxazin-6-yl)-1,3,5-triazinane-2,4-dione (CAS 1258836-72-4/trifludimoxazin), at least one and especially exactly one herbicidally active compound from group b13), in particular selected from the group consisting of 2,4-D and its salts and esters, aminocyclopyrachlor and its salts and esters, aminopyralid and its salts such as aminopyralid-tris(2-hydroxypropyl)ammonium and its esters, clopyralid and its salts and esters, dicamba and its salts and esters, fluroxypyr-meptyl, quinclorac and quinmerac.

According to another preferred embodiment of the invention, the composition comprises, in addition to a PPO-inhibiting herbicide, preferably acifluorfen, acifluorfen-sodium, butafenacil, cinidon-ethyl, carfentrazone-ethyl, flumioxazin, fluthiacet-methyl, fomesafen, lactofen, oxadiargyl, oxyfluorfen, saflufenacil, sulfentrazone, ethyl [3-[2-chloro-4-fluoro-5-(1-methyl-6-trifluoromethyl-2,4-dioxo-1,2,3,4-tetrahydropyrimidin-3-yl)phenoxy]-2-pyridyloxy]acetate (CAS 353292-31-6; S-3100), 1,5-dimethyl-6-thioxo-3-(2,2,7-trifluoro-3-oxo-4-(prop-2-ynyl)-3,4-dihydro-2H-benzo[b][1,4]oxazin-6-yl)-1,3,5-triazinane-2,4-dione (CAS 1258836-72-4/trifludimoxazin), especially preferred saflufenacil, 1,5-dimethyl-6-thioxo-3-(2,2,7-trifluoro-3-oxo-4-(prop-2-ynyl)-3,4-dihydro-2H-benzo[b][1,4]oxazin-6-yl)-1,3,5-triazinane-2,4-dione (CAS 1258836-72-4/trifludimoxazin), at least one and especially exactly one herbicidally active compound from group b14), in particular selected from the group consisting of diflufenzopyr and diflufenzopyr-sodium.

According to another preferred embodiment of the invention, the composition comprises, in addition to a PPO-inhibiting herbicide, preferably acifluorfen, acifluorfen-sodium, butafenacil, cinidon-ethyl, carfentrazone-ethyl, flumioxazin, fluthiacet-methyl, fomesafen, lactofen, oxadiargyl, oxyfluorfen, saflufenacil, sulfentrazone, ethyl [3-[2-chloro-4-fluoro-5-(1-methyl-6-trifluoromethyl-2,4-dioxo-1,2,3,4-tetrahydropyrimidin-3-yl)phenoxy]-2-pyridyloxy]acetate (CAS 353292-31-6; S-3100), 1,5-dimethyl-6-thioxo-3-(2,2,7-trifluoro-3-oxo-4-(prop-2-ynyl)-3,4-dihydro-2H-benzo[b][1,4]oxazin-6-yl)-1,3,5-triazinane-2,4-dione (CAS 1258836-72-4/trifludimoxazin), especially preferred saflufenacil, 1,5-dimethyl-6-thioxo-3-(2,2,7-trifluoro-3-oxo-4-(prop-2-ynyl)-3,4-dihydro-2H-benzo[b][1,4]oxazin-6-yl)-1,3,5-triazinane-2,4-dione (CAS 1258836-72-4/trifludimoxazin), at least one and especially exactly one herbicidally active compound from group b15), in particular selected from the group consisting of dymron (=daimuron), indanofan, indaziflam, oxaziclomefone and triaziflam.

Here and below, the term “binary compositions” includes compositions comprising one or more, for example 1, 2 or 3, active compounds of the PPO-inhibiting herbicide and either one or more, for example 1, 2 or 3, herbicides B.

In binary compositions comprising at least one PPO-inhibiting herbicide as component A and at least one herbicide B, the weight ratio of the active compounds A:B is generally in the range of from 1:1000 to 1000:1, preferably in the range of from 1:500 to 500:1, in particular in the range of from 1:250 to 250:1 and particularly preferably in the range of from 1:75 to 75:1.

Particularly preferred herbicides B are the herbicides B as defined above; in particular the herbicides B.1-B.229 listed below in table B:

TABLE B Herbicide B B.1 clethodim B.2 clodinafop-propargyl B.3 cycloxydim B.4 cyhalofop-butyl B.5 fenoxaprop-ethyl B.6 fenoxaprop-P-ethyl B.7 fluazifop B.8 metamifop B.9 pinoxaden B.10 profoxydim B.11 quizalofop B.12 sethoxydim B.13 tepraloxydim B.14 tralkoxydim B.15 esprocarb B.16 ethofumesate B.17 molinate B.18 prosulfocarb B.19 thiobencarb B.20 triallate B.21 bensulfuron-methyl B.22 bispyribac-sodium B.23 cloransulam-methyl B.24 chlorsulfuron B.25 clorimuron B.26 cyclosulfamuron B.27 diclosulam B.28 florasulam B.29 flumetsulam B.30 flupyrsulfuron-methyl-sodium B.31 foramsulfuron B.32 halosulfuron-methyl B.33 imazamox B.34 imazamox-ammonium B.35 imazapic B.36 imazapic-ammonium B.37 imazapic-isopropylammonium B.38 imazapyr B.39 imazapyr-ammonium B.40 imazapyr-isopropylammonium B.41 imazaquin B.42 imazaquin-ammonium B.43 imazethapyr B.44 imazethapyr-ammonium B.45 imazethapyr-isopropylammonium B.46 imazosulfuron B.47 iodosulfuron-methyl-sodium B.48 iofensulfuron B.49 iofensulfuron-sodium B.50 mesosulfuron-methyl B.51 metazosulfuron B.52 metsulfuron-methyl B.53 metosulam B.54 nicosulfuron B.55 penoxsulam B.56 propoxycarbazon-sodium B.57 pyrazosulfuron-ethyl B.58 pyribenzoxim B.59 pyriftalid B.60 pyrithiobac-sodium B.61 pyroxsulam B.62 propyrisulfuron B.63 rimsulfuron B.64 sulfosulfuron B.65 thiencarbazone-methyl B.66 thifensulfuron-methyl B.67 tribenuron-methyl B.68 trifloxysulfuron B.69 tritosulfuron B.70 triafamone B.71 ametryne B.72 atrazine B.73 bentazon B.74 bromoxynil B.75 bromoxynil-octanoate B.76 bromoxynil-heptanoate B.77 bromoxynil-potassium B.78 diuron B.79 fluometuron B.80 hexazinone B.81 isoproturon B.82 linuron B.83 metamitron B.84 metribuzin B.85 prometryne B.86 propanil B.87 simazin B.88 terbuthylazine B.89 terbutryn B.90 paraquat-dichloride B.91 acifluorfen B.92 acifluorfen-sodium B.93 azafenidin B.94 bencarbazone B.95 benzfendizone B.96 bifenox B.97 butafenacil B.98 carfentrazone B.99 carfentrazone-ethyl B.100 chlomethoxyfen B.101 cinidon-ethyl B.102 fluazolate B.103 flufenpyr B.104 flufenpyr-ethyl B.105 flumiclorac B.106 flumiclorac-pentyl B.107 flumioxazin B.108 fluoroglycofen B.109 fluoroglycofen-ethyl B.110 fluthiacet B.111 fluthiacet-methyl B.112 fomesafen B.113 halosafen B.114 lactofen B.115 oxadiargyl B.116 oxadiazon B.117 oxyfluorfen B.118 pentoxazone B.119 profluazol B.120 pyraclonil B.121 pyraflufen B.122 pyraflufen-ethyl B.123 saflufenacil B.124 sulfentrazone B.125 thidiazimin B.126 tiafenacil B.127 ethyl [3-[2-chloro-4-fluoro-5- (1-methyl-6-trifluoromethyl- 2,4-dioxo-1,2,3,4-tetrahydro- pyrimidin-3-yl)phenoxy]-2- pyridyloxy]acetate (CAS 353292-31-6) B.128 1,5-dimethyl-6-thioxo-3-(2,2,7- trifluoro-3-oxo-4-(prop-2- ynyl)-3,4-dihydro-2H- benzo[b][1,4]oxazin-6-yl)-1,3,5- triazinane-2,4-dione (CAS 1258836-72-4/ trifludimoxazin) B.129 N-ethyl-3-(2,6-dichloro-4- trifluoromethylphenoxy)-5- methyl-1H-pyrazole-1- carboxamide (CAS 452098-92-9) B.130 N-tetrahydrofurfuryl-3-(2,6- dichloro-4- trifluoromethylphenoxy)-5- methyl-1H-pyrazole-1- carboxamide (CAS 915396-43-9) B.131 N-ethyl-3-(2-chloro-6-fluoro- 4-trifluoromethylphenoxy)-5- methyl-1H-pyrazole-1- carboxamide (CAS 452099-05-7) B.132 N-tetrahydrofurfuryl-3-(2- chloro-6-fluoro-4-trifluoro- methylphenoxy)-5-methyl-1H- pyrazole-1-carboxamide (CAS 452100-03-7) B.133 3-[7-fluoro-3-oxo-4-(prop-2- ynyl)-3,4-dihydro-2H- benzo[1,4]oxazin-6-yl]-1,5- dimethyl-6-thioxo- [1,3,5]triazinan-2,4-dione B.134 2-(2,2,7-Trifluoro-3-oxo-4- prop-2-ynyl-3,4-dihydro-2H- benzo[1,4]oxazin-6-yl)-4,5,6,7- tetrahydro-isoindole-1,3-dione B.135 1-Methyl-6-trifluoromethyl-3- (2,2,7-trifluoro-3-oxo-4-prop- 2-ynyl-3,4-dihydro-2H- benzo[1,4]oxazin-6-yl)-1H- pyrimidine-2,4-dione B.136 methyl (E)-4-[2-chloro-5-[4- chloro-5-(difluoromethoxy)- 1H-methyl-pyrazol-3-yl]-4- fluoro-phenoxy]-3-methoxy- but-2-enoate [CAS 948893- 00-3] B.137 3-[7-Chloro-5-fluoro-2- (trifluoromethyl)-1H- benzimidazol-4-yl]-1-methyl- 6-(trifluoromethyl)-1H- pyrimidine-2,4-dione (CAS 212754-02-4) B.138 benzobicyclon B.139 clomazone B.140 diflufenican B.141 flurochloridone B.142 isoxaflutole B.143 mesotrione B.144 norflurazone B.145 picolinafen B.146 sulcotrione B.147 tefuryltrione B.148 tembotrione B.149 topramezone B.150 topramezone-sodium B.151 bicyclopyrone B.152 amitrole B.153 fluometuron B.154 glyphosate B.155 glyphosate-ammonium B.156 glyphosate-dimethylammonium B.157 glyphosate-isopropylammonium B.158 glyphosate-trimesium (sulfosate) B.159 glyphosate-potassium B.160 glufosinate B.161 glufosinate-ammonium B.162 glufosinate-P B.163 glufosinate-P-ammonium B.164 pendimethalin B.165 trifluralin B.166 acetochlor B.167 butachlor B.168 cafenstrole B.169 dimethenamid-P B.170 fentrazamide B.171 flufenacet B.172 mefenacet B.173 metazachlor B.174 metolachlor B.175 S-metolachlor B.176 pretilachlor B.177 fenoxasulfone B.178 isoxaben B.179 ipfencarbazone B.180 pyroxasulfone B.181 2,4-D B.182 2,4-D-isobutyl B.183 2,4-D-dimethylammonium B.184 2,4-D-N,N,N-trimethylethanolammonium B.185 aminopyralid B.186 aminopyralid-methyl B.187 aminopyralid-tris(2-hydroxypropyl)ammonium B.188 clopyralid B.189 clopyralid-methyl B.190 clopyralid-olamine B.191 dicamba B.192 dicamba-butotyl B.193 dicamba-diglycolamine B.194 dicamba-dimethylammonium B.195 dicamba-diolamine B.196 dicamba-isopropylammonium B.197 dicamba-potassium B.198 dicamba-sodium B.199 dicamba-trolamine B.200 dicamba-N,N-bis-(3-aminopropyl)methylamine B.201 dicamba-diethylenetriamine B.202 fluroxypyr B.203 fluroxypyr-meptyl B.204 MCPA B.205 MCPA-2-ethylhexyl B.206 MCPA-dimethylammonium B.207 quinclorac B.208 quinclorac-dimethylammonium B.209 quinmerac B.210 quinmerac-dimethylammonium B.211 aminocyclopyrachlor B.212 aminocyclopyrachlor-potassium B.213 aminocyclopyrachlor-methyl B.214 diflufenzopyr B.215 diflufenzopyr-sodium B.216 dymron B.217 indanofan B.218 indaziflam B.219 oxaziclomefone B.220 triaziflam B.221 II.1 B.222 II.2 B.223 II.3 B.224 II.4 B.225 II.5 B.226 II.6 B.227 II.7 B.228 II.8 B.229 II.9

Particularly preferred are compositions 1.1 to 1.229, comprising acifluorfen and the substance(s) as defined in the respective row of table B-1:

TABLE B-1 (compositions 1.1 to 1.229): comp. no. herbicide B 1.1 B.1 1.2 B.2 1.3 B.3 1.4 B.4 1.5 B.5 1.6 B.6 1.7 B.7 1.8 B.8 1.9 B.9 1.10 B.10 1.11 B.11 1.12 B.12 1.13 B.13 1.14 B.14 1.15 B.15 1.16 B.16 1.17 B.17 1.18 B.18 1.19 B.19 1.20 B.20 1.21 B.21 1.22 B.22 1.23 B.23 1.24 B.24 1.25 B.25 1.26 B.26 1.27 B.27 1.28 B.28 1.29 B.29 1.30 B.30 1.31 B.31 1.32 B.32 1.33 B.33 1.34 B.34 1.35 B.35 1.36 B.36 1.37 B.37 1.38 B.38 1.39 B.39 1.40 B.40 1.41 B.41 1.42 B.42 1.43 B.43 1.44 B.44 1.45 B.45 1.46 B.46 1.47 B.47 1.48 B.48 1.49 B.49 1.50 B.50 1.51 B.51 1.52 B.52 1.53 B.53 1.54 B.54 1.55 B.55 1.56 B.56 1.57 B.57 1.58 B.58. 1.59 B.59 1.60 B.60 1.61 B.61 1.62 B.62 1.63 B.63 1.64 B.64 1.65 B.65 1.66 B.66 1.67 B.67 1.68 B.68 1.69 B.69 1.70 B.70 1.71 B.71 1.72 B.72 1.73 B.73 1.74 B.74 1.75 B.75 1.76 B.76 1.77 B.77 1.78 B.78 1.79 B.79 1.80 B.80 1.81 B.81 1.82 B.82 1.83 B.83 1.84 B.84 1.85 B.85 1.86 B.86 1.87 B.87 1.88 B.88 1.89 B.89 1.90 B.90 1.91 B.91 1.92 B.92 1.93 B.93 1.94 B.94 1.95 B.95 1.96 B.96 1.97 B.97 1.98 B.98 1.99 B.99 1.100 B.100 1.101 B.101 1.102 B.102 1.103 B.103 1.104 B.104 1.105 B.105 1.106 B.106 1.107 B.107 1.108 B.108 1.109 B.109 1.110 B.110 1.111 B.111 1.112 B.112 1.113 B.113 1.114 B.114 1.115 B.115 1.116 B.116 1.117 B.117 1.118 B.118 1.119 B.119 1.120 B.120 1.121 B.121 1.122 B.122 1.123 B.123 1.124 B.124 1.125 B.125 1.126 B.126 1.127 B.127 1.128 B.128 1.129 B.129 1.130 B.130 1.131 B.131 1.132 B.132 1.133 B.133 1.134 B.134 1.135 B.135 1.136 B.136 1.137 B.137 1.138 B.138 1.139 B.139 1.140 B.140 1.141 B.141 1.142 B.142 1.143 B.143 1.144 B.144 1.145 B.145 1.146 B.146 1.147 B.147 1.148 B.148 1.149 B.149 1.150 B.150 1.151 B.151 1.152 B.152 1.153 B.153 1.154 B.154 1.155 B.155 1.156 B.156 1.157 B.157 1.158 B.158 1.159 B.159 1.160 B.160 1.161 B.161 1.162 B.162 1.163 B.163 1.164 B.164 1.165 B.165 1.166 B.166 1.167 B.167 1.168 B.168 1.169 B.169 1.170 B.170 1.171 B.171 1.172 B.172 1.173 B.173 1.174 B.174 1.175 B.175 1.176 B.176 1.177 B.177 1.178 B.178 1.179 B.179 1.180 B.180 1.181 B.181 1.182 B.182 1.183 B.183 1.184 B.184 1.185 B.185 1.186 B.186 1.187 B.187 1.188 B.188 1.189 B.189 1.190 B.190 1.191 B.191 1.192 B.192 1.193 B.193 1.194 B.194 1.195 B.195 1.196 B.196 1.197 B.197 1.198 B.198 1.199 B.199 1.200 B.200 1.201 B.201 1.202 B.202 1.203 B.203 1.204 B.204 1.205 B.205 1.206 B.206 1.207 B.207 1.208 B.208 1.209 B.209 1.210 B.210 1.211 B.211 1.212 B.212 1.213 B.213 1.214 B.214 1.215 B.215 1.216 B.216 1.217 B.217 1.218 B.218 1.219 B.219 1.220 B.220 1.221 B.221 1.222 B.222 1.223 B.223 1.224 B.224 1.225 B.225 1.226 B.226 1.227 B.227 1.228 B.228 1.229 B.229

Also especially preferred are compositions 2.1. to 2.229 which differ from the corresponding compositions 1.1 to 1.229 only in that they comprise as component A acifluorfen-sodium.

Also especially preferred are compositions 3.1. to 3.229 which differ from the corresponding compositions 1.1 to 1.229 only in that they comprise as component A azafenidin.

Also especially preferred are compositions 4.1. to 4.229 which differ from the corresponding compositions 1.1 to 1.229 only in that they comprise as component A bencarbazone.

Also especially preferred are compositions 5.1. to 5.229 which differ from the corresponding compositions 1.1 to 1.229 only in that they comprise as component A benzfendizone.

Also especially preferred are compositions 6.1. to 6.229 which differ from the corresponding compositions 1.1 to 1.229 only in that they comprise as component A bifenox.

Also especially preferred are compositions 7.1. to 7.229 which differ from the corresponding compositions 1.1 to 1.227 only in that they comprise as component A butafenacil.

Also especially preferred are compositions 8.1. to 8.229 which differ from the corresponding compositions 1.1 to 1.229 only in that they comprise as component A carfentrazone.

Also especially preferred are compositions 9.1. to 9.229which differ from the corresponding compositions 1.1 to 1. 229 only in that they comprise as component A carfentrazone-ethyl.

Also especially preferred are compositions 10.1. to 10.229 which differ from the corresponding compositions 1.1 to 1.229 only in that they comprise as component A chlomethoxyfen.

Also especially preferred are compositions 11.1. to 11.229 which differ from the corresponding compositions 1.1 to 1.229 only in that they comprise as component A cinidon-ethyl.

Also especially preferred are compositions 12.1. to 12.229 which differ from the corresponding compositions 1.1 to 1.229 only in that they comprise as component A fluazolate.

Also especially preferred are compositions 13.1. to 13.229 which differ from the corresponding compositions 1.1 to 1.229 only in that they comprise as component A flufenpyr.

Also especially preferred are compositions 14.1. to 14.229 which differ from the corresponding compositions 1.1 to 1.229 only in that they comprise as component A flufenpyr-ethyl.

Also especially preferred are compositions 15.1. to 15.229 which differ from the corresponding compositions 1.1 to 1.229 only in that they comprise as component A flumiclorac.

Also especially preferred are compositions 16.1. to 16.229 which differ from the corresponding compositions 1.1 to 1.229 only in that they comprise as component A flumiclorac-pentyl.

Also especially preferred are compositions 17.1. to 17.229 which differ from the corresponding compositions 1.1 to 1.229 only in that they comprise as component A flumioxazin.

Also especially preferred are compositions 18.1. to 18.229 which differ from the corresponding compositions 1.1 to 1.229 only in that they comprise as component A fluoroglycofen.

Also especially preferred are compositions 19.1. to 19.229 which differ from the corresponding compositions 1.1 to 1.229 only in that they comprise as component A fluoroglycofen-ethyl.

Also especially preferred are compositions 20.1. to 20.229 which differ from the corresponding compositions 1.1 to 1.229 only in that they comprise as component A fluthiacet.

Also especially preferred are compositions 21.1. to 21.229 which differ from the corresponding compositions 1.1 to 1.229 only in that they comprise as component A fluthiacet-methyl.

Also especially preferred are compositions 22.1. to 22.229 which differ from the corresponding compositions 1.1 to 1.229 only in that they comprise as component A fomesafen.

Also especially preferred are compositions 23.1. to 23.229 which differ from the corresponding compositions 1.1 to 1.229 only in that they comprise as component A halosafen.

Also especially preferred are compositions 24.1. to 24.229 which differ from the corresponding compositions 1.1 to 1.229 only in that they comprise as component A lactofen.

Also especially preferred are compositions 25.1. to 25.229 which differ from the corresponding compositions 1.1 to 1.229 only in that they comprise as component A oxadiargyl.

Also especially preferred are compositions 26.1. to 26.229 which differ from the corresponding compositions 1.1 to 1.229 only in that they comprise as component A oxadiazon.

Also especially preferred are compositions 27.1. to 27.229 which differ from the corresponding compositions 1.1 to 1.229 only in that they comprise as component A oxyfluorfen.

Also especially preferred are compositions 28.1. to 28.229 which differ from the corresponding compositions 1.1 to 1.229 only in that they comprise as component A pentoxazone.

Also especially preferred are compositions 29.1. to 29.229 which differ from the corresponding compositions 1.1 to 1.229 only in that they comprise as component A profluazol.

Also especially preferred are compositions 30.1. to 30.229 which differ from the corresponding compositions 1.1 to 1.229 only in that they comprise as component A pyraclonil.

Also especially preferred are compositions 31.1. to 31.229 which differ from the corresponding compositions 1.1 to 1.229 only in that they comprise as component A pyraflufen.

Also especially preferred are compositions 32.1. to 32.229 which differ from the corresponding compositions 1.1 to 1.229 only in that they comprise as component A pyraflufen-ethyl.

Also especially preferred are compositions 33.1. to 33.229 which differ from the corresponding compositions 1.1 to 1.229 only in that they comprise as component A saflufenacil.

Also especially preferred are compositions 34.1. to 34.229 which differ from the corresponding compositions 1.1 to 1.229 only in that they comprise as component A sulfentrazone.

Also especially preferred are compositions 35.1. to 35.229 which differ from the corresponding compositions 1.1 to 1.229 only in that they comprise as component A thidiazimin.

Also especially preferred are compositions 36.1. to 36.229 which differ from the corresponding compositions 1.1 to 1.229 only in that they comprise as component A tiafenacil.

Also especially preferred are compositions 37.1. to 37.229 which differ from the corresponding compositions 1.1 to 1.229 only in that they comprise as component A ethyl [3-[2-chloro-4-fluoro-5-(1-methyl-6-trifluoromethyl-2,4-dioxo-1,2,3,4-tetrahydropyrimidin-3-yl)phenoxy]-2-pyridyloxy]acetate (CAS 353292-31-6; S-3100).

Also especially preferred are compositions 38.1. to 38.229 which differ from the corresponding compositions 1.1 to 1.229 only in that they comprise as component A 1,5-dimethyl-6-thioxo-3-(2,2,7-trifluoro-3-oxo-4-(prop-2-ynyl)-3,4-dihydro-2H-benzo[b][1,4]oxazin-6-yl)-1,3,5-triazinane-2,4-dione (CAS 1258836-72-4/trifludimoxazin)

Also especially preferred are compositions 39.1. to 39.229 which differ from the corresponding compositions 1.1 to 1.229 only in that they comprise as component A N-ethyl-3-(2,6-dichloro-4-trifluoromethylphenoy)-5-methyl-1H-pyrazole-1-carboxamide (CAS 452098-92-9).

Also especially preferred are compositions 40.1. to 40.229 which differ from the corresponding compositions 1.1 to 1.229 only in that they comprise as component A N-tetrahydrofurfuryl-3-(2,6-dichloro-4-trifluoromethylphenoxy)-5-methyl-1H-pyrazole-1-carboxamide (CAS 915396-43-9).

Also especially preferred are compositions 41.1. to 41.229 which differ from the corresponding compositions 1.1 to 1.229 only in that they comprise as component A

N-ethyl-3-(2-chloro-6-fluoro-4-trifluoromethylphenoxy)-5-methyl-1H-pyrazole-1-carboxamide (CAS 452099-05-7).

Also especially preferred are compositions 42.1. to 42.229 which differ from the corresponding compositions 1.1 to 1.229 only in that they comprise as component A N-tetrahydrofurfuryl-3-(2-chloro-6-fluoro-4-trifluoromethylphenoxy)-5-methyl-1H-pyrazole-1-carboxamide (CAS 452100-03-7).

Also especially preferred are compositions 43.1. to 43.229 which differ from the corresponding compositions 1.1 to 1.229 only in that they comprise as component A 3-[7-fluoro-3-oxo-4-(prop-2-ynyl)-3,4-dihydro-2H-benzo[1,4]oxazin-6-yl]-1,5-dimethyl-6-thioxo-[1,3,5]triazinan-2,4-dione.

Also especially preferred are compositions 44.1. to 44.229 which differ from the corresponding compositions 1.1 to 1.229 only in that they comprise as component A methyl (E)-4-[2-chloro-5-[4-chloro-5-(difluoromethoxy)-1H-methyl-pyrazol-3-yl]-4-fluoro-phenoxy]-3-methoxy-but-2-enoate (CAS 948893-00-3).

Also especially preferred are compositions 45.1. to 45.229 which differ from the corresponding compositions 1.1 to 1.229 only in that they comprise as component A 3-[7-Chloro-5-fluoro-2-(trifluoromethyl)-1H-benzimidazol-4-yl]-1-methyl-6-(trifluoromethyl)-1H-pyrimidine-2,4-dione (CAS 212754-02-4).

Also especially preferred are compositions 46.1. to 46.229 which differ from the corresponding compositions 1.1 to 1.229 only in that they comprise as component A 2-(2,2,7-Trifluoro-3-oxo-4-prop-2-ynyl-3,4-dihydro-2H-benzo[1,4]oxazin-6-yl)-4,5,6,7-tetrahydro-isoindole-1,3-dione.

Also especially preferred are compositions 47.1. to 47.229 which differ from the corresponding compositions 1.1 to 1.229 only in that they comprise as component A 1-Methyl-6-trifluoromethyl-3-(2,2,7-trifluoro-3-oxo-4-prop-2-ynyl-3,4-dihydro-2H-benzo[1,4]oxazin-6-yl)-1H-pyrimidine-2,4-dione

Also especially preferred are compositions 48.1. to 48.229 which differ from the corresponding compositions 1.1 to 1.229 only in that they additionally comprise benoxacor as safener C.

Also especially preferred are compositions 49.1. to 49.229 which differ from the corresponding compositions 1.1 to 1.229 only in that they additionally comprise cloquintocet as safener C.

Also especially preferred are compositions 50.1. to 50.229 which differ from the corresponding compositions 1.1 to 1.229 only in that they additionally comprise cyprosulfamide as safener C.

Also especially preferred are compositions 51.1. to 51.229 which differ from the corresponding compositions 1.1 to 1.229 only in that they additionally comprise dichlormid as safener C.

Also especially preferred are compositions 52.1. to 52.229 which differ from the corresponding compositions 1.1 to 1.229 only in that they additionally comprise fenchlorazole as safener C.

Also especially preferred are compositions 53.1. to 53.229 which differ from the corresponding compositions 1.1 to 1.229 only in that they additionally comprise fenclorim as safener C.

Also especially preferred are compositions 54.1. to 54.229 which differ from the corresponding compositions 1.1 to 1.229 only in that they additionally comprise furilazole as safener C.

Also especially preferred are compositions 55.1. to 55.229 which differ from the corresponding compositions 1.1 to 1.229 only in that they additionally comprise isoxadifen as safener C.

Also especially preferred are compositions 56.1. to 56.229 which differ from the corresponding compositions 1.1 to 1.229 only in that they additionally comprise mefenpyr as safener C.

Also especially preferred are compositions 57.1. to 57.229 which differ from the corresponding compositions 1.1 to 1.229 only in that they additionally comprise 4-(dichloroacetyl)-1-oxa-4-azaspiro[4.5]decane (MON4660, CAS 71526-07-3) as safener C.

Also especially preferred are compositions 58.1. to 58.229 which differ from the corresponding compositions 1.1 to 1.229 only in that they additionally comprise 2,2,5-trimethyl-3-(dichloroacetyl)-1,3-oxazolidine (R-29148, CAS 52836-31-4) as safener C.

It is generally preferred to use the compounds of the invention in combination with herbicides that are selective for the crop being treated and which complement the spectrum of weeds controlled by these compounds at the application rate employed. It is further generally preferred to apply the compounds of the invention and other complementary herbicides at the same time, either as a combination formulation or as a tank mix.

In another embodiment, the present invention refers to a method for identifying a herbicide by using a mutated PPO encoded by a nucleic acid which comprises the nucleotide sequence of SEQ ID NO: 118, 119, 120, 121, 122, 123, 124, 125, 126, 127, 128, or 130, or a variant or derivative thereof.

Said method comprises the steps of:

-   a) generating a transgenic cell or plant comprising a nucleic acid     encoding a mutated PPO, wherein the mutated PPO is expressed; -   b) applying a herbicide to the transgenic cell or plant of a) and to     a control cell or plant of the same variety; -   c) determining the growth or the viability of the transgenic cell or     plant and the control cell or plant after application of said     herbicide, and -   d) selecting “ herbicides” which confer reduced growth to the     control cell or plant as compared to the growth of the transgenic     cell or plant.

As described above, the present invention teaches compositions and methods for increasing the tolerance of a crop plant or seed as compared to a wild-type variety of the plant or seed. In a preferred embodiment, the tolerance of a crop plant or seed is increased such that the plant or seed can withstand a herbicide application of preferably approximately 1-1000 g ai ha⁻¹, more preferably 1-200 g ai ha⁻¹, even more preferably 5-150 g ai ha⁻¹, and most preferably 10-100 g ai ha⁻¹. As used herein, to “withstand” a herbicide application means that the plant is either not killed or only moderately injured by such application. It will be understood by the person skilled in the art that the application rates may vary, depending on the environmental conditions such as temperature or humidity, and depending on the chosen kind of herbicide (active ingredient ai).

Post-emergent weed control methods useful in various embodiments hereof utilize about >0.3× application rates of herbicides; in some embodiments, this can be about, for example, >0.3×, >0.4×, >0.5×, >0.6×, >0.7×, >0.8×, >0.9×, or >1× of herbicides. In one embodiment, herbicide-tolerant plants of the present invention have tolerance to a post-emergant application of a herbicides at an amount of about 25 to about 200 g ai/ha. In some embodiments, wherein the herbicide-tolerant plant is a dicot (e.g., soy, cotton), the post-emergant application of the herbicides is at an amount of about 50 g ai/ha. In another embodiment, wherein the herbicide-tolerant plant is a monocot (e.g., maize, rice, sorghum), the post-emergant application of the herbicides is at an amount of about 200 g ai/ha. In other embodiments, wherein the herbicide-tolerant plant is a Brassica (e.g., canola), the post-emergant application of the herbicides is at an amount of about 25 g ai/ha. In post-emergent weed control methods hereof, in some embodiments, the method can utilize herbicides application rates at about 7 to 10 days post-emergent. In another embodiment, the application rate can exceed 1× herbicides; in some embodiments, the rate can be up to 4× herbicides, though more typically it will be about 2.5× or less, or about 2× or less, or about 1× or less.

Furthermore, the present invention provides methods that involve the use of at least one herbicide, optionally in combination with one or more herbicidal compounds B, and, optionally, a safener C, as described in detail supra.

In these methods, the herbicide can be applied by any method known in the art including, but not limited to, seed treatment, soil treatment, and foliar treatment. Prior to application, the herbicide can be converted into the customary formulations, for example solutions, emulsions, suspensions, dusts, powders, pastes and granules. The use form depends on the particular intended purpose; in each case, it should ensure a fine and even distribution of the compound according to the invention.

By providing plants having increased tolerance to herbicide, a wide variety of formulations can be employed for protecting plants from weeds, so as to enhance plant growth and reduce competition for nutrients. A herbicide can be used by itself for pre-emergence, post-emergence, pre-planting, and at-planting control of weeds in areas surrounding the crop plants described herein, or a herbicide formulation can be used that contains other additives. The herbicide can also be used as a seed treatment. Additives found in a herbicide formulation include other herbicides, detergents, adjuvants, spreading agents, sticking agents, stabilizing agents, or the like. The herbicide formulation can be a wet or dry preparation and can include, but is not limited to, flowable powders, emulsifiable concentrates, and liquid concentrates. The herbicide and herbicide formulations can be applied in accordance with conventional methods, for example, by spraying, irrigation, dusting, or the like.

Suitable formulations are described in detail in PCT/EP2009/063387 and PCT/EP2009/063386, which are incorporated herein by reference.

As disclosed herein, the PPO nucleic acids of the invention find use in enhancing the herbicide tolerance of plants that comprise in their genomes a gene encoding a herbicide-tolerant mutated PPO protein. Such a gene may be an endogenous gene or a transgene, as described above. Additionally, in certain embodiments, the nucleic acids of the present invention can be stacked with any combination of polynucleotide sequences of interest in order to create plants with a desired phenotype. For example, the nucleic acids of the present invention may be stacked with any other polynucleotides encoding polypeptides having pesticidal and/or insecticidal activity, such as, for example, the Bacillus thuringiensis toxin proteins (described in U.S. Pat. Nos. 5,366,892; 5,747,450; 5,737,514; 5,723,756; 5,593,881; and Geiser et al (1986) Gene 48: 109), 5-enolpyruvylshikimate-3-phosphate synthase (EPSPS), Glyphosate acetyl transferase (GAT), cytochrome P450 monooxygenase, phosphinothricin acetyltransferase (PAT), Acetohydroxyacid synthase (AHAS; EC 4.1.3.18, also known as acetolactate synthase or ALS), hydroxyphenyl pyruvate dioxygenase (HPPD), Phytoene desaturase (PD), Protoporphyrinogen oxidase (PPO) and dicamba degrading enzymes as disclosed in WO 02/068607, or phenoxyaceticacid- and phenoxypropionicacid-derivative degrading enzymes as disclosed in WO 2008141154 or WO 2005107437. The combinations generated can also include multiple copies of any one of the polynucleotides of interest.

Consequently, Herbicide-tolerant plants of the invention can be used in conjunction with an herbicide to which they are tolerant. Herbicides can be applied to the plants of the invention using any techniques known to those skilled in the art. Herbicides can be applied at any point in the plant cultivation process. For example, herbicides can be applied pre-planting, at planting, pre-emergence, post-emergence or combinations thereof. Herbicides may be applied to seeds and dried to form a layer on the seeds.

In some embodiments, seeds are treated with a safener, followed by a post-emergent application of a herbicides. In one embodiment, the post-emergent application of the herbicides is about 7 to 10 days following planting of safener-treated seeds. In some embodiments, the safener is cloquintocet, dichlormid, fluxofenim, or combinations thereof.

Methods of controlling weeds or undesired vegetation

In other aspects, the present invention provides a method for controlling weeds at a locus for growth of a plant or plant part thereof, the method comprising: applying a composition comprising a herbicides to the locus.

In some aspects, the present invention provides a method for controlling weeds at a locus for growth of a plant, the method comprising: applying an herbicide composition comprising herbicides to the locus; wherein said locus is: (a) a locus that contains: a plant or a seed capable of producing said plant; or (b) a locus that is to be after said applying is made to contain the plant or the seed; wherein the plant or the seed comprises in at least some of its cells a polynucleotide operably linked to a promoter operable in plant cells, the promoter capable of expressing a mutated PPO polypeptide encoded by the polynucleotide, the expression of the mutated PPO polypeptide conferring to the plant tolerance to herbicides.

Herbicide compositions hereof can be applied, e.g., as foliar treatments, soil treatments, seed treatments, or soil drenches. Application can be made, e.g., by spraying, dusting, broadcasting, or any other mode known useful in the art.

In one embodiment, herbicides can be used to control the growth of weeds that may be found growing in the vicinity of the herbicide-tolerant plants invention. In embodiments of this type, an herbicide can be applied to a plot in which herbicide-tolerant plants of the invention are growing in vicinity to weeds. An herbicide to which the herbicide-tolerant plant of the invention is tolerant can then be applied to the plot at a concentration sufficient to kill or inhibit the growth of the weed. Concentrations of herbicide sufficient to kill or inhibit the growth of weeds are known in the art and are disclosed above.

In other embodiments, the present invention provides a method for controlling weeds in the vicinity of a herbicide-tolerant plant of the invention. The method comprises applying an effective amount of a herbicides to the weeds and to the auxinic herbicide-tolerant plant, wherein the plant has increased tolerance to auxinic herbicide when compared to a wild-type plant. In some embodiments, the herbicide-tolerant plants of the invention are preferably crop plants, including, but not limited to, sunflower, alfalfa, Brassica sp., soybean, cotton, safflower, peanut, tobacco, tomato, potato, wheat, rice, maize, sorghum, barley, rye, millet, and sorghum.

In other aspects, herbicide(s) (e.g., herbicides) can also be used as a seed treatment. In some embodiments, an effective concentration or an effective amount of herbicide(s), or a composition comprising an effective concentration or an effective amount of herbicide(s) can be applied directly to the seeds prior to or during the sowing of the seeds. Seed Treatment formulations may additionally comprise binders and optionally colorants.

Binders can be added to improve the adhesion of the active materials on the seeds after treatment. In one embodiments, suitable binders are block copolymers EO/PO surfactants but also polyvinylalcoholsl, polyvinylpyrrolidones, polyacrylates, polymethacrylates, polybutenes, polyisobutylenes, polystyrene, polyethyleneamines, polyethyleneamides, polyethyleneimines (Lupasol®, Polymin®), polyethers, polyurethans, polyvinylacetate, tylose and copolymers derived from these polymers. Optionally, also colorants can be included in the formulation. Suitable colorants or dyes for seed treatment formulations are Rhodamin B, C.I. Pigment Red 112, C.I. Solvent Red 1, pigment blue 15:4, pigment blue 15:3, pigment blue 15:2, pigment blue 15: 1, pigment blue 80, pigment yellow 1, pigment yellow 13, pigment red 112, pigment red 48:2, pigment red 48: 1, pigment red 57: 1, pigment red 53:1, pigment orange 43, pigment orange 34, pigment orange 5, pigment green 36, pigment green 7, pigment white 6, pigment brown 25, basic violet 10, basic violet 49, acid red 51, acid red 52, acid red 14, acid blue 9, acid yellow 23, basic red 10, basic red 108.

The term seed treatment comprises all suitable seed treatment techniques known in the art, such as seed dressing, seed coating, seed dusting, seed soaking, and seed pelleting. In one embodiment, the present invention provides a method of treating soil by the application, in particular into the seed drill: either of a granular formulation containing the herbicides as a composition/formulation (e.g., a granular formulation), with optionally one or more solid or liquid, agriculturally acceptable carriers and/or optionally with one or more agriculturally acceptable surfactants. This method is advantageously employed, for example, in seedbeds of cereals, maize, cotton, and sunflower.

The present invention also comprises seeds coated with or containing with a seed treatment formulation comprising herbicides and at least one other herbicide such as, e.g. , an AHAS-inhibitor selected from the group consisting of amidosulfuron, azimsulfuron, bensulfuron, chlorimuron, chlorsulfuron, cinosulfuron, cyclosulfamuron, ethametsulfuron, ethoxysulfuron, flazasulfuron, flupyrsulfuron, foramsulfuron, halosulfuron, imazosulfuron, iodosulfuron, mesosulfuron, metsulfuron, nicosulfuron, oxasulfuron, primisulfuron, prosulfuron, pyrazosulfuron, rimsulfuron, sulfometuron, sulfosulfuron, thifensulfuron, triasulfuron, tribenuron, trifloxysulfuron, triflusulfuron, tritosulfuron, imazamethabenz, imazamox, imazapic, imazapyr, imazaquin, imazethapyr, cloransulam, diclosulam, florasulam, flumetsulam, metosulam, penoxsulam, bispyribac, pyriminobac, propoxycarbazone, flucarbazone, pyribenzoxim, pyriftalid and pyrithiobac.

The term “coated with and/or containing” generally signifies that the active ingredient is for the most part on the surface of the propagation product at the time of application, although a greater or lesser part of the ingredient may penetrate into the propagation product, depending on the method of application. When the said propagation product is (re)planted, it may absorb the active ingredient.

In some embodiments, the seed treatment application with herbicides or with a formulation comprising the herbicides is carried out by spraying or dusting the seeds before sowing of the plants and before emergence of the plants.

In other embodiments, in the treatment of seeds, the corresponding formulations are applied by treating the seeds with an effective amount of herbicides or a formulation comprising the herbicides.

In other aspects, the present invention provides a method for combating undesired vegetation or controlling weeds comprising contacting the seeds of the herbicide-tolerant plants of the present invention before sowing and/or after pregermination with herbicides. The method can further comprise sowing the seeds, for example, in soil in a field or in a potting medium in greenhouse. The method finds particular use in combating undesired vegetation or controlling weeds in the immediate vicinity of the seed. The control of undesired vegetation is understood as the killing of weeds and/or otherwise retarding or inhibiting the normal growth of the weeds. Weeds, in the broadest sense, are understood as meaning all those plants which grow in locations where they are undesired.

The weeds of the present invention include, for example, dicotyledonous and monocotyledonous weeds. Dicotyledonous weeds include, but are not limited to, weeds of the genera: Sinapis, Lepiclium, Galium, Stellaria, Matricaria, Anthemis, Galinsoga, Chenopodium, Urtica, Senecio, Amaranthus, Portulaca, Xanthium, Convolvulus, Ipomoea, Polygonum, Sesbania, Ambrosia, Cirsium, Carduus, Sonchus, Solarium, Rorippa, Rotala, Lindernia, Lamium, Veronica, Abutilon, Emex, Datura, Viola, Galeopsis, Papaver, Centaurea, Trifolium, Ranunculus, and Taraxacum. Monocotyledonous weeds include, but are not limited to, weeds of the genera: Echinochloa, Setaria, Panicum, Digitaria, Phleum, Poa, Festuca, Eleusine, Brachiaria, Lolium, Bromus, Avena, Cyperus, Sorghum, Agropyron, Cynodon, Monochoria, Fimbristyslis, Sagittaria, Eleocharis, Scirpus, Paspalum, Ischaemum, Sphenoclea, Dactyloctenium, Agrostis, Alopecurus, and Apera.

In addition, the weeds of the present invention can include, for example, crop plants that are growing in an undesired location. For example, a volunteer maize plant that is in a field that predominantly comprises soybean plants can be considered a weed, if the maize plant is undesired in the field of soybean plants.

In other embodiments, in the treatment of seeds, the corresponding formulations are applied by treating the seeds with an effective amount of herbicides or a formulation comprising the herbicides.

In still further aspects, treatment of loci, plants, plant parts, or seeds of the present invention comprises application of an agronomically acceptable composition that does not contain an A.I. In one embodiment, the treatment comprises application of an agronomically acceptable composition that does not contain a herbicides A.I. In some embodiments, the treatment comprises application of an agronomically acceptable composition that does not contain a herbicides A.L, wherein the composition comprises one or more of agronomically-acceptable carriers, diluents, excipients, plant growth regulators, and the like. In other embodiments, the treatment comprises application of an agronomically acceptable composition that does not contain a herbicides A.I., wherein the composition comprises an adjuvant. In one embodiment, the adjuvant is a surfactant, a spreader, a sticker, a penetrant, a drift-control agent, a crop oil, an emulsifier, a compatibility agent, or combinations thereof.

It should also be understood that the foregoing relates to preferred embodiments of the present invention and that numerous changes may be made therein without departing from the scope of the invention. The invention is further illustrated by the following examples, which are not to be construed in any way as imposing limitations upon the scope thereof. On the contrary, it is to be clearly understood that resort may be had to various other embodiments, modifications, and equivalents thereof, which, after reading the description herein, may suggest themselves to those skilled in the art without departing from the spirit of the present invention and/or the scope of the appended claims.

EXAMPLES

Legend to PPO-inhibitors referring to Uracilpyridines which can be used in the following examples:

Uracilpyridine 1 ethyl 2-[[3-[2-chloro-4-fluoro-5-[3-methyl-2,6-dioxo- 4-(trifluoromethyl)pyrimidin-1-yl]phenoxy]-2- pyridyl]oxy]acetate Uracilpyridine 2 ethyl 2-[[3-[[3-chloro-5-fluoro-6-[3-methyl-2,6-dioxo- 4-(trifluoromethyl)pyrimidin-1-yl]-2-pyridyl]oxy]-2- pyridyl]oxy]acetate Uracilpyridine 3 2-[[3-[[3-chloro-5-fluoro-6-[3-methyl-2,6-dioxo- 4-(trifluoromethyl)pyrimidin-1-yl]-2-pyridyl]oxy]-2- pyridyl]oxy]acetic acid Uracilpyridine 4 ethyl 2-[2-[[3-chloro-5-fluoro-6-[3-methyl-2,6-dioxo- 4-(trifluoromethyl)pyrimidin-1-yl]-2- pyridyl]oxy]phenoxy]acetate Uracilpyridine 5 2-[2-[[3-chloro-5-fluoro-6-[3-methyl-2,6-dioxo- 4-(trifluoromethyl)pyrimidin-1-yl]-2- pyridyl]oxy]phenoxy]acetic acid Uracilpyridine 6 ethyl 2-[2-[[3-bromo-6-[3-methyl-2,6-dioxo- 4-(trifluoromethyl)pyrimidin-1-yl]-2- pyridyl]oxy]phenoxy]acetate Uracilpyridine 7 ethyl 2-[2-[[3-chloro-5-fluoro-6-[3-methyl-2,6-dioxo- 4-(trifluoromethyl)pyrimidin-1-yl]-2- pyridyl]oxy]-4-fluoro-phenoxy]acetate Uracilpyridine 8 ethyl 2-[2-[[3,5-difluoro-6-[3-methyl-2,6-dioxo- 4-(trifluoromethyl)pyrimidin-1-yl]-2- pyridyl]oxy]phenoxy]acetate Uracilpyridine 9 2-[2-[[3,5-difluoro-6-[3-methyl-2,6-dioxo- 4-(trifluoromethyl)pyrimidin-1-yl]-2- pyridyl]oxy]phenoxy]acetic acid Uracilpyridine 10 2-[2-[[3-chloro-5-fluoro-6-[3-methyl-2,6-dioxo- 4-(trifluoromethyl)pyrimidin-1-yl]-2- pyridyl]oxy]phenoxy]-N-methylsulfonyl-acetamide Uracilpyridine 11 ethyl 2-[[3-[[3-chloro-6-[3,5-dimethyl-2,6-dioxo- 4-(trifluoromethyl)pyrimidin-1-yl]-5-fluoro-2- pyridyl]oxy]-2-pyridyl]oxy]acetate Uracilpyridine 12 ethyl 2-[2-[[3-chloro-6-[3,5-dimethyl-2,6-dioxo- 4-(trifluoromethyl)pyrimidin-1-yl]-5-fluoro-2- pyridyl]oxy]phenoxy]acetate Uracilpyridine 13 allyl 2-[[3-[[3-chloro-5-fluoro-6-[3-methyl-2,6-dioxo- 4-(trifluoromethyl)pyrimidin-1-yl]-2-pyridyl]oxy]-2- pyridyl]oxy]acetate Uracilpyridine 14 prop-2-ynyl 2-[[3-[[3-chloro-5-fluoro-6-[3- methyl-2,6-dioxo-4-(trifluoromethyl)pyrimidin-1- yl]-2-pyridyl]oxy]-2-pyridyl]oxy]acetate Uracilpyridine 15 cyclopropylmethyl 2-[[3-[[3-chloro-5-fluoro-6-[3- methyl-2,6-dioxo-4-(trifluoromethyl)pyrimidin-1- yl]-2-pyridyl]oxy]-2-pyridyl]oxy]acetate Uracilpyridine 16 2,2-difluoroethyl 2-[[3-[[3-chloro-5-fluoro-6-[3- methyl-2,6-dioxo-4-(trifluoromethyl)pyrimidin-1-yl]-2- pyridyl]oxy]-2-pyridyl]oxy]acetate Uracilpyridine 17 isobutyl 2-[[3-[[3-chloro-5-fluoro-6-[3-methyl-2,6- dioxo-4-(trifluoromethyl)pyrimidin-1-yl]-2- pyridyl]oxy]-2-pyridyl]oxy]acetate Uracilpyridine 18 (2-ethoxy-2-oxo-ethyl) 2-[[3-[[3-chloro-5-fluoro-6-[3- methyl-2,6-dioxo-4-(trifluoromethyl)pyrimidin-1- yl]-2-pyridyl]oxy]-2-pyridyl]oxy]acetate Uracilpyridine 19 2-methoxyethyl 2-[[3-[[3-chloro-5-fluoro-6-[3-methyl- 2,6-dioxo-4-(trifluoromethyl)pyrimidin-1-yl]-2- pyridyl]oxy]-2-pyridyl]oxy]acetate

Example 1 Site-Directed Mutagenesis PPO

All nucleic acid coding sequence and all single and double mutants encoding a polypeptide comprising SEQ ID NO: 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, 100, 101, 102, 103, 104, 105, 106, 107, 108, 109, 110, 111, 112, 113, 114, 115, 116, 117, or 129, are synthesized and cloned by Geneart (Geneart AG, Regensburg, Germany). Rational design mutants are synthesized by Geneart. Random PPO gene libraries are synthesized by Geneart. Plasmids are isolated from E co/iTOP10 by performing a plasmid minpreparation and confirmed by DNA sequencing.

Example 2 Expression and Purification of Recombinant Wildtype and Mutant PPO

(Taken from: Franck E. Dayan, Pankaj R. Daga, Stephen O. Duke, Ryan M. Lee, Patrick J. Tranel, Robert J. Doerksen. Biochemical and structural consequences of a glycine deletion in the α-8 helix of protoporphyrinogen oxidase. Biochimica et Biophysica Acta 1804 (2010), 1548-56) Clones in pRSET vector are transformed into BL21(DE3)-pLysS strain of E. coli Cells are grown in 250 mL of LB with 100 μgmL−1 of carbenicillin, shaking overnight at 37° C. Cultures are diluted in 1 L of LB with antibiotic and grown at 37° C. shaking for 2 h, induced with 1 mM IPTG and grown at 25° C. shaking for 5 more hours. The cells are harvested by centrifugation at 1600×g, washed with 0.09% NaCl, and stored at −80° C. Cells are lysed using a French press at 140 MPa in 50 mM sodium phosphate pH 7.5, 1 M NaCl, 5 mM imidazole, 5% glycerol, and 1 μg mL−1 leupeptin. Following lysis, 0.5 U of benzonase (Novagen, EMD Chemicals, Inc., Gibbstown, N.J.) and PMSF (final concentration of 1 mM) are added. Cell debris is removed by centrifugation at 3000×g. His-tagged PPO proteins are purified on a nickel activated Hitrap Chelating HP column (GE Healthcare Bio-Sciences Corp., Piscataway, N.J.) equilibrated with 20 mM sodium phosphate pH 8.0, 50 mM NaCl, 5 mM imidazole, 5 mM MgCl2, 0.1 mM EDTA, and 17% glycerol. PPO is eluted with 250 mM imidazole. The active protein is desalted on a PD-10 column (GE Healthcare Bio-Sciences Corp., Piscataway, N.J.) equilibrated with a 20 mM sodium phosphate buffer, pH 7.5, 5 mM MgCl2, 1 mM EDTA and 17% glycerol. Each litre of culture provided approximately 10 mg of pure PPO, which is stored at −20° C. until being used in assays.

Example 3: PPO Enzyme Assay (Non-Recombinant)

PPO protein (EC 1.3.3.4) is extracted from coleoptiles or shoots (150 g fresh weight) of dark-grown corn, black nightshade, morning glory, and velvetleaf seedlings as described previously (Grossmann et al. 2010). Before harvesting, the seedlings are allowed to green for 2 hours in the light in order to achieve the highest specific enzyme activities in the thylakoid fractions at low chlorophyll concentrations. At high chlorophyll concentrations significant quenching of fluorescence occurs, which limits the amount of green thylakoids that can be used in the test. Plant materials are homogenized in the cold with a Braun blender using a fresh-weight-to-volume ratio of 1:4. Homogenization buffer consisted of tris(hydroxymethyl)aminomethane (Tris)-HCl (50 mM; pH 7.3), sucrose (0.5 M), magnesium chloride (1 mM), ethylenediaminetetraacetic acid (EDTA) (1 mM) and bovine serum albumin (2 g L⁻¹). After filtration through four layers of Miracloth, crude plastid preparations are obtained after centrifugation at 10 000×g for 5 min and resuspension in homogenization buffer before centrifugation at 150×g for 2 min to remove crude cell debris. The supernatant is centrifuged at 4000×g for 15 min and the pellet fraction is resuspended in 1 ml of a buffer containing Tris-HCl (50 mM; pH 7.3), EDTA (2 mM), leupeptin (2 μM), pepstatin (2 μM) and glycerol (200 ml L⁻¹) and stored at −80° C. until use. Protein is determined in the enzyme extract with bovine serum albumin as a standard. PPO activity is assayed fluorometrically by monitoring the rate of Proto formation from chemically reduced protoporphyrinogen IX under initial velocity conditions. The assay mixture consisted of Tris-HCl (100 mM; pH 7.3), EDTA (1 mM), dithiothreitol (5 mM), Tween 80 (0.085%), protoporphyrinogen IX (2 μM), and 40 μg extracted protein in a total volume of 200 μl. The reaction is initiated by addition of substrate protoporphyrinogen IX at 22° C. saflufenacil, 1,5-dimethyl-6-thioxo-3-(2,2,7-trifluoro-3-oxo-4-(prop-2-ynyl)-3,4-dihydro-2H-benzo[b][1,4]oxazin-6-yl)-1,3,5-triazinane-2,4-dione (CAS 1258836-72-4/trifludimoxazin), flumioxazin, butafenacil, acifluorfen, lactofen, bifenox, sulfentrazone, and photosynthesis inhibitor diuron as negative control are prepared in dimethyl sulfoxide (DMSO) solution (0.1 mM concentration of DMSO in the assay) and added to the assay mixture in concentrations of 0.005 pM to 5 μM before incubation. Fluorescence is monitored directly from the assay mixture using a POLARstar Optima/Galaxy (BMG) with excitation at 405 nm and emission monitored at 630 nm. Non-enzymatic activity in the presence of heat-inactivated extract is negligible. Inhibition of enzyme activity induced by the herbicide is expressed as percentage inhibition relative to untreated controls. Molar concentrations of compound required for 50% enzyme inhibition (IC₅₀ values) are calculated by fitting the values to the dose-response equation using non-linear regression analysis.

Example 4 PPO Enzyme Assay (Recombinant)

Proto is purchased from Sigma-Aldrich (Milwaukee, Wis.). Protogen is prepared according to Jacobs and Jacobs (N. J. Jacobs, J. M. Jacobs, Assay for enzymatic protoporphyrinogen oxidation, a late step in heme synthesis, Enzyme 28 (1982) 206-219). Assays are conducted in 100 mM sodium phosphate pH 7.4 with 0.1 mM EDTA, 0.1% Tween 20, 5 μM FAD, and 500 mM imidazole. Dose—response curves with the PPO inhibitors saflufenacil, 1,5-dimethyl-6-thioxo-3-(2,2,7-trifluoro-3-oxo-4-(prop-2-ynyl)-3,4-dihydro-2H-benzo[b][1,4]oxazin-6-yl)-1,3,5-triazinane-2,4-dione (CAS 1258836-72-4/trifludimoxazin), flumioxazin, butafenacil, acifluorfen, lactofen, bifenox, sulfentrazone, and photosynthesis inhibitor diuron as negative control, and MC-15608 are obtained in the presence of 150 μM Protogen. The excitation and emission bandwidths are set at 1.5 and 30 nm, respectively. All assays are made in duplicates or triplicates and measured using a POLARstar Optima/Galaxy (BMG) with excitation at 405 nm and emission monitored at 630 nm. Molar concentrations of compound required for 50% enzyme inhibition (IC₅₀ values) are calculated by fitting the values to the dose-response equation using non-linear regression analysis. The results are shown in Table x.

Normalized SEQ. Activity ID (FU · Saflufenacil Trifludimoxazine Saflufenacil Trifludimoxazine Amino Acid Substitution NO. min⁻¹ · ng⁻¹) IC50 (M) Tolerance Factor PPO herbicide sensitive 1 40.00 1.86E−09 5.17E−10 1.00 1.00 PPO2 WC PPO herbicide sensitive PPO2 AC 2 32.00 1.78E−10 5.96E−11 0.10 0.12 dG210 3 & 4 3.20 1.60E−06 2.12E−09 860.26 4.09 R128L 1 & 2 28.00 2.22E−07 7.73E−10 119.60 1.50 R128A, G211N, F420M 1 & 2 0.33 1.00E−05 2.91E−07 5376.63 562.54 G211D, F420M 1 & 2 0.40 3.38E−07 1.32E−07 181.98 254.58 R128A, G211V, F420M 1 & 2 0.49 1.00E−05 1.15E−07 5376.63 222.66 R128A, G211A, F420V 1 & 2 0.62 1.00E−05 9.31E−08 5376.63 180.06 R128A, G2105, F420M 1 & 2 0.40 1.00E−05 6.83E−08 5376.63 132.20 G211N, F420M 1 & 2 0.56 1.00E−05 3.55E−08 5376.63 68.62 R128A, S412N, F420M 1 & 2 42.00 1.00E−05 2.43E−08 5376.63 46.93 Q119L, R128A, F420M 1 & 2 25.83 1.00E−05 2.06E−08 5376.63 39.94 R128A, G211C, F420M 1 & 2 12.49 1.00E−05 1.97E−08 5376.63 38.16 R128A, G211A, F420M 1 & 2 6.53 1.00E−05 1.52E−08 5376.63 29.49 G211A, L397Q, F420M 1 & 2 2.60 1.00E−05 1.09E−08 5376.63 21.13 G211C, F420V 1 & 2 4.23 7.67E−06 6.28E−09 4125.38 12.14 G211A, F420V 1 & 2 3.65 4.12E−06 5.46E−09 2215.82 10.57 R128V, G211V 1 & 2 0.58 6.60E−06 2.91E−09 3546.09 5.64 G210S, F420M 1 & 2 2.22 1.00E−05 2.03E−09 5376.63 3.93 G210S, F420M 1 & 2 2.13 1.00E−05 2.03E−09 5376.63 3.93 G211A, F420M 1 & 2 35.95 2.76E−06 1.51E−09 1481.85 2.93 G210C, F420M 1 & 2 5.66 2.58E−06 1.44E−09 1385.04 2.78 R128A, G210S 1 & 2 0.56 1.00E−05 1.34E−09 5376.63 2.60 G211R 1 & 2 1.20 1.13E−08 1.22E−09 6.09 2.36 G211V, F420M 1 & 2 5.09 7.89E−06 1.21E−09 4240.14 2.33 G211W 1 & 2 0.76 2.68E−08 1.10E−09 14.41 2.13 M414Q, F420M 1 & 2 17.20 2.80E−06 1.09E−09 1504.86 2.10 G210T 1 & 2 1.24 1.06E−06 1.00E−09 567.63 1.94 Q119L, F420M 1 & 2 46.86 1.20E−06 9.78E−10 644.80 1.89 G211C, F420M 1 & 2 9.30 2.32E−06 8.62E−10 1246.07 1.67 F420M, d, V527 1 & 2 18.89 1.19E−06 8.15E−10 638.14 1.58 G211D 1 & 2 0.80 2.54E−08 7.53E−10 13.66 1.46 G211L 1 & 2 0.66 4.27E−07 7.42E−10 229.47 1.44 d_K528 1 & 2 73.43 7.33E−10 1.42 G211P 1 & 2 0.62 1.41E−08 7.01E−10 7.58 1.36 G211A 1 & 2 16.81 3.86E−09 6.87E−10 2.08 1.33 G210S 1 & 2 5.90 5.18E−08 6.39E−10 27.87 1.24 d_M529 1 & 2 70.49 6.31E−10 1.22 S387Y, F420M 1 & 2 9.28 9.92E−07 6.26E−10 533.18 1.21 D372E, F420M 1 & 2 20.82 2.40E−06 6.21E−10 1287.85 1.20 G211N 1 & 2 6.00 7.47E−09 6.05E−10 4.02 1.17 S412N, F420M 1 & 2 36.54 1.51E−06 6.01E−10 813.36 1.16 G211K 1 & 2 6.25 2.43E−09 5.91E−10 1.30 1.14 E436I, F420M 1 & 2 33.28 3.06E−06 5.78E−10 1644.41 1.12 E436I, F420M 1 & 2 33.29 3.06E−06 5.78E−10 1644.41 1.12 M343T, F420M 1 & 2 25.24 2.77E−06 5.52E−10 1489.90 1.07 R128A, G211A 1 & 2 11.92 3.71E−07 5.39E−10 199.39 1.04 d_K532 1 & 2 76.24 5.33E−10 1.03 G211F 1 & 2 0.81 1.79E−08 5.00E−10 9.64 0.97 Q447K 1 & 2 21.00 8.82E−10 4.93E−10 0.47 0.95 d_V527 1 & 2 74.52 4.40E−10 0.85 G211I 1 & 2 0.95 1.05E−08 4.35E−10 5.63 0.84 Q119L, R128A, Q323H, 1 & 2 86.12 2.12E−07 4.00E−10 114.19 0.77 S412N G211V 1 & 2 11.69 4.23E−09 3.99E−10 2.28 0.77 G211S 1 & 2 2.86 2.58E−08 3.94E−10 13.87 0.76 G211A, L397Q 1 & 2 2.33 5.63E−07 3.88E−10 302.70 0.75 d_D530 1 & 2 69.56 3.61E−10 0.70 d_E531 1 & 2 77.64 3.52E−10 0.68 S387L 1 & 2 7.38 6.78E−09 3.03E−10 3.64 0.59 S412T 1 & 2 33.24 6.44E−10 2.89E−10 0.35 0.56 G210S, G211A 1 & 2 3.25 4.36E−08 2.70E−10 23.43 0.52 G211T 1 & 2 1.63 9.98E−08 2.67E−10 53.65 0.52 S387K 1 & 2 11.21 6.21E−09 2.57E−10 3.34 0.50 Q119L, Q323H, S412N, 1 & 2 51.44 1.41E−06 2.55E−10 759.42 0.49 F420M R128A, G211C 1 & 2 4.28 3.57E−07 2.49E−10 191.74 0.48 d_T533 1 & 2 76.72 2.39E−10 0.46 d_A534 1 & 2 64.01 2.34E−10 0.45 S412L 1 & 2 51.35 4.54E−10 2.27E−10 0.24 0.44 S412M 1 & 2 78.96 4.46E−10 2.25E−10 0.24 0.43 G211M 1 & 2 5.25 3.11E−09 2.22E−10 1.67 0.43 S412V 1 & 2 50.22 7.60E−10 2.16E−10 0.41 0.42 Q119L, Q323H, S412N 1 & 2 52.00 8.24E−10 2.05E−10 0.44 0.40 Q323H, S412N 1 & 2 58.70 4.79E−10 1.96E−10 0.26 0.38 S412Y 1 & 2 43.26 5.39E−10 1.91E−10 0.29 0.37 S412D 1 & 2 70.61 5.92E−10 1.85E−10 0.32 0.36 S412P 1 & 2 40.06 6.66E−10 1.82E−10 0.36 0.35 d_S523 1 & 2 56.26 1.73E−10 0.33 S412Q 1 & 2 28.47 5.25E−10 1.72E−10 0.28 0.33 S412K 1 & 2 95.26 3.10E−10 1.64E−10 0.17 0.32 M343T 1 & 2 73.85 1.23E−09 1.64E−10 0.66 0.32 d_I525 1 & 2 58.17 1.59E−10 0.31 T451V 1 & 2 72.24 1.01E−09 1.59E−10 0.54 0.31 G211H 1 & 2 1.78 2.94E−09 1.57E−10 1.58 0.30 S412A 1 & 2 27.00 4.53E−10 1.53E−10 0.24 0.30 Q119L, R128A 1 & 2 77.43 1.82E−07 1.52E−10 97.59 0.29 S412E 1 & 2 65.94 5.92E−10 1.51E−10 0.32 0.29 N431G 1 & 2 53.81 7.68E−10 1.47E−10 0.41 0.28 E436I 1 & 2 49.37 1.13E−09 1.47E−10 0.61 0.28 S412R 1 & 2 30.41 6.97E−10 1.44E−10 0.38 0.28 Q446G 1 & 2 71.53 7.40E−10 1.40E−10 0.40 0.27 S412I 1 & 2 89.83 5.16E−10 1.40E−10 0.28 0.27 d_Y526 1 & 2 40.19 1.39E−10 0.27 d_H524 1 & 2 66.29 1.34E−10 0.26 S412H 1 & 2 83.20 4.98E−10 1.33E−10 0.27 0.26 R335S 1 & 2 58.65 8.02E−10 1.21E−10 0.43 0.23 G210C, G211A 1 & 2 2.78 5.80E−08 1.16E−10 31.16 0.22 S433P 1 & 2 58.76 6.87E−10 1.14E−10 0.37 0.22 F345G 1 & 2 43.65 7.17E−10 1.14E−10 0.39 0.22 d_D522 1 & 2 29.16 1.13E−10 0.22 D435S 1 & 2 39.66 8.39E−10 1.13E−10 0.45 0.22 K428D 1 & 2 65.87 6.17E−10 1.11E−10 0.33 0.22 G210C, G211C 1 & 2 1.07 4.42E−08 1.10E−10 23.75 0.21 M414Q 1 & 2 42.49 1.15E−09 1.04E−10 0.62 0.20 G210C 1 & 2 2.08 4.74E−08 9.93E−11 25.50 0.19 S412W 1 & 2 21.68 5.82E−10 9.86E−11 0.31 0.19 S412F 1 & 2 33.89 3.21E−10 8.76E−11 0.17 0.17 T358D 1 & 2 54.96 9.66E−10 8.33E−11 0.52 0.16 G210T, G211A 1 & 2 0.61 3.67E−06 8.33E−11 1970.69 0.16 S464R 1 & 2 49.36 5.35E−10 8.11E−11 0.29 0.16 D372E 1 & 2 46.86 1.02E−09 8.02E−11 0.55 0.16 S412G 1 & 2 49.90 4.15E−10 7.95E−11 0.22 0.15 R128A, S412N 1 & 2 55.34 1.89E−07 7.45E−11 101.43 0.14 R425H 1 & 2 21.44 7.71E−10 7.15E−11 0.41 0.14 R128A, G120C 1 & 2 3.77 2.85E−06 7.15E−11 1532.53 0.14 G210S, G211C 1 & 2 0.86 2.09E−08 6.99E−11 11.21 0.14 S412C 1 & 2 19.62 2.69E−10 6.70E−11 0.14 0.13 S412N 1 & 2 15.00 1.01E−08 6.69E−11 5.43 0.13 Q119L, S412N 1 & 2 48.64 6.43E−10 6.13E−11 0.35 0.12 K373D 1 & 2 26.17 8.76E−10 4.64E−11 0.47 0.09 S387Y 1 & 2 17.02 1.27E−09 4.51E−11 0.68 0.09 E356K 1 & 2 19.90 7.12E−10 4.40E−11 0.38 0.09 i_Q391 1 & 2 14.30 7.99E−10 3.32E−11 0.43 0.06 D372E, K373D 1 & 2 14.21 5.78E−10 2.92E−11 0.31 0.06 D372E, K373D 1 & 2 40.00 5.78E−10 2.92E−11 0.31 0.06 C415H 1 & 2 20.35 8.15E−10 2.52E−11 0.44 0.05 M414Q, E436I 1 & 2 32.72 1.19E−09 0.64 0.00 R128A, G211A, S387L, F420M 1 & 2 51.20 1.00E−05 5376.63 G210T, G211C 1 & 2 0.50 8.97E−06 4823.04 Q119T, G211A, F420M 1 & 2 38.68 3.49E−06 1878.54 S387L, F420M 1 & 2 10.44 2.95E−06 1584.85 G210C, L397Q 1 & 2 0.22 2.47E−06 1326.31 G210C, G211V 1 & 2 0.18 2.13E−06 1144.05 G211A, S387L, F420M 1 & 2 115.08 1.99E−06 1069.44 Q119T, F420M 1 & 2 56.44 7.90E−07 424.49 R128A, S387L 1 & 2 53.92 3.25E−07 174.56 K127N, R128C 1 & 2 12.16 3.09E−08 16.63 S387V 1 & 2 3.84 1.76E−08 2.06E−10 9.48 S387V, R425N 1 & 2 5.00 1.54E−08 8.29 Y129N 1 & 2 6.16 7.20E−09 3.87 Y129F 1 & 2 45.06 5.57E−09 2.99 Q119T 1 & 2 12.52 5.46E−09 2.93 Q119I 1 & 2 6.61 4.68E−09 2.52 M343I 1 & 2 21.00 4.29E−09 2.31 S387T 1 & 2 18.25 4.22E−09 2.27 S387I 1 & 2 5.33 4.11E−09 2.21 M414W 1 & 2 9.56 4.09E−09 2.20 D372S 1 & 2 10.39 4.07E−09 2.19 d_P40 1 & 2 38.64 3.93E−09 2.11 G211A, S387L 1 & 2 38.68 3.58E−09 1.92 0372N 1 & 2 18.56 3.53E−09 1.90 G264A 1 & 2 23.60 3.42E−09 1.84 E436M 1 & 2 6.85 3.28E−09 1.76 Q323P 1 & 2 42.67 3.25E−09 1.75 M414D 1 & 2 12.70 3.20E−09 1.72 S387L, M414Q 1 & 2 0.00 3.13E−09 1.68 Q119T, M414Q 1 & 2 17.52 3.06E−09 1.65 S387D 1 & 2 33.37 3.00E−09 1.61 Q119H 1 & 2 4.52 2.98E−09 1.60 M414R 1 & 2 7.32 2.91E−09 1.56 M414A 1 & 2 3.16 2.83E−09 1.52 D286G 1 & 2 27.60 2.79E−09 1.50 i_Q391, H392K, N393H 1 & 2 36.90 2.78E−09 1.49 D372K 1 & 2 22.19 2.77E−09 1.49 E436A 1 & 2 21.31 2.73E−09 1.47 M343K 1 & 2 23.30 2.66E−09 1.43 S387N 1 & 2 4.57 2.65E−09 1.42 K248G 1 & 2 52.40 2.65E−09 1.42 D372F 1 & 2 15.16 2.58E−09 1.39 S387F 1 & 2 9.56 2.51E−09 1.35 Q119A 1 & 2 8.60 2.51E−09 1.35 M343A 1 & 2 46.40 2.49E−09 1.34 M414V 1 & 2 24.60 2.44E−09 1.31 E436W 1 & 2 14.87 2.43E−09 1.31 M343F 1 & 2 64.97 2.41E−09 1.30 S387C 1 & 2 28.97 2.38E−09 1.28 M343R 1 & 2 38.24 2.38E−09 1.28 E436Y 1 & 2 22.32 2.34E−09 1.26 D372V 1 & 2 22.28 2.25E−09 1.21 S387L, S412N, E436I 1 & 2 10.80 2.25E−09 1.21 D372M 1 & 2 11.96 2.23E−09 1.20 S412N, E436I 1 & 2 55.60 2.19E−09 1.18 D372Y 1 & 2 29.55 2.19E−09 1.18 M414Y 1 & 2 41.86 2.18E−09 1.17 M343D 1 & 2 109.09 2.17E−09 1.16 S387M 1 & 2 17.37 2.15E−09 1.16 N126H 1 & 2 43.52 2.11E−09 1.13 M414S 1 & 2 9.98 2.10E−09 1.13 M414H 1 & 2 18.84 2.09E−09 1.13 M343G 1 & 2 57.77 2.07E−09 1.11 D372Q 1 & 2 27.27 2.07E−09 1.11 M343L 1 & 2 16.36 2.06E−09 1.11 E436V 1 & 2 25.92 2.06E−09 1.11 Q323K 1 & 2 11.36 2.06E−09 1.11 P260R 1 & 2 38.88 2.04E−09 1.10 D372P 1 & 2 18.90 2.04E−09 1.10 M414N 1 & 2 5.76 2.02E−09 1.09 M343S 1 & 2 40.54 1.96E−09 1.05 E436F 1 & 2 10.80 1.95E−09 1.05 E436G 1 & 2 10.02 1.94E−09 1.04 Q323V 1 & 2 37.40 1.94E−09 1.04 Q323Y 1 & 2 16.92 1.94E−09 1.04 D372T 1 & 2 32.59 1.94E−09 1.04 S387H 1 & 2 24.32 1.93E−09 1.04 L400F 1 & 2 42.92 1.93E−09 1.04 Q323N 1 & 2 17.72 1.91E−09 1.03 M414K 1 & 2 10.50 1.88E−09 1.01 M414I 1 & 2 7.40 1.88E−09 1.01 E436S 1 & 2 19.88 1.88E−09 1.01 A104L 1 & 2 37.88 1.86E−09 1.00 Q119W 1 & 2 2.84 1.86E−09 1.00 S108R 1 & 2 41.22 1.85E−09 0.99 V53I 1 & 2 28.30 1.83E−09 0.99 M343V 1 & 2 24.66 1.83E−09 0.98 H65S 1 & 2 25.72 1.79E−09 0.96 Q323S 1 & 2 15.60 1.79E−09 0.96 Q323D 1 & 2 97.37 1.78E−09 0.96 D372G 1 & 2 25.12 1.75E−09 0.94 E436K 1 & 2 12.71 1.72E−09 0.93 S387G 1 & 2 12.56 1.72E−09 0.93 S387A 1 & 2 20.13 1.68E−09 0.90 G308D 1 & 2 16.52 1.65E−09 0.89 E436D 1 & 2 48.58 1.64E−09 0.88 Q323M 1 & 2 10.27 1.64E−09 0.88 L139I 1 & 2 38.44 1.64E−09 0.88 G211A, M414Q 1 & 2 60.72 1.61E−09 0.86 Q323I 1 & 2 37.28 1.60E−09 0.86 K61R 1 & 2 40.92 1.59E−09 0.86 Q323E 1 & 2 20.85 1.53E−09 0.82 G210A 1 & 2 1.96 1.51E−09 3.34E−11 0.81 E249D 1 & 2 32.36 1.51E−09 0.81 Q119V 1 & 2 24.43 1.49E−09 0.80 Q119S 1 & 2 3.24 1.49E−09 0.80 K87D 1 & 2 26.40 1.48E−09 0.79 L71V 1 & 2 29.80 1.47E−09 0.79 S64K 1 & 2 36.44 1.46E−09 0.78 D372A 1 & 2 23.84 1.44E−09 0.78 D372R 1 & 2 34.71 1.44E−09 0.77 R116E 1 & 2 8.74 1.42E−09 0.76 S387L, E436I 1 & 2 11.12 1.41E−09 0.76 V262K 1 & 2 14.76 1.41E−09 0.76 Q323T 1 & 2 33.63 1.40E−09 0.75 Q323W 1 & 2 26.80 1.39E−09 0.75 Q323G 1 & 2 33.71 1.38E−09 0.74 Q119C 1 & 2 16.32 1.38E−09 0.74 S412N, M414Q 1 & 2 53.92 1.37E−09 0.74 E436N 1 & 2 23.80 1.36E−09 0.73 G32A 1 & 2 37.88 1.35E−09 0.72 d_K250 1 & 2 49.52 1.35E−09 0.72 M343Q 1 & 2 33.34 1.32E−09 0.71 Q119T, M414Q, E436I 1 & 2 16.44 1.31E−09 0.70 D372I 1 & 2 15.58 1.29E−09 0.69 Q119T, G211A, S387L 1 & 2 9.84 1.29E−09 0.69 G252K 1 & 2 20.80 1.28E−09 0.69 I257G 1 & 2 39.08 1.28E−09 0.69 D372L 1 & 2 7.59 1.27E−09 0.68 D372H 1 & 2 20.08 1.27E−09 0.68 A255V 1 & 2 53.62 1.26E−09 0.68 D453G 1 & 2 56.62 1.26E−09 0.68 I91L 1 & 2 35.48 1.24E−09 0.66 L245T 1 & 2 39.72 1.22E−09 0.66 D482G 1 & 2 23.58 1.21E−09 0.65 S387L, S412N 1 & 2 6.72 1.20E−09 0.65 Q323C 1 & 2 19.95 1.20E−09 0.65 M414L 1 & 2 17.36 1.20E−09 0.64 Q119R 1 & 2 4.29 1.20E−09 0.64 M434C 1 & 2 33.31 1.19E−09 0.64 M343W 1 & 2 30.46 1.19E−09 0.64 E436H 1 & 2 24.43 1.18E−09 0.64 S387W 1 & 2 3.31 1.16E−09 0.62 N309G 1 & 2 40.04 1.15E−09 0.62 K259G 1 & 2 45.70 1.14E−09 0.61 E436L 1 & 2 9.44 1.13E−09 0.61 S246A 1 & 2 40.08 1.12E−09 0.60 K127F 1 & 2 11.84 1.12E−09 0.60 Q119T, S412N, E436I 1 & 2 30.88 1.12E−09 0.60 Q119T, S412N 1 & 2 41.72 1.11E−09 0.60 Q119M 1 & 2 41.27 1.11E−09 0.60 E253G 1 & 2 37.22 1.10E−09 0.59 S387E 1 & 2 58.75 1.10E−09 0.59 Q119T, S387L, E436I 1 & 2 4.68 1.10E−09 0.59 D88S 1 & 2 24.50 1.10E−09 0.59 Q119T, E436I 1 & 2 64.80 1.09E−09 0.59 L67V 1 & 2 34.12 1.07E−09 0.57 N254S 1 & 2 59.68 1.06E−09 0.57 V85N 1 & 2 26.94 1.05E−09 0.57 Q119Y 1 & 2 8.07 1.05E−09 0.57 P305S 1 & 2 50.58 1.05E−09 0.56 E436R 1 & 2 21.81 1.03E−09 0.55 M414F 1 & 2 30.06 1.02E−09 0.55 M343H 1 & 2 30.84 1.02E−09 0.55 K83R 1 & 2 17.24 1.01E−09 0.54 D372W 1 & 2 17.84 1.00E−09 0.54 M343Y 1 & 2 9.02 9.87E−10 0.53 E436C 1 & 2 19.62 9.74E−10 0.52 Q323F 1 & 2 22.61 9.52E−10 0.51 K127N 1 & 2 8.20 9.37E−10 0.50 E436T 1 & 2 15.79 9.35E−10 0.50 M343N 1 & 2 38.49 9.29E−10 0.50 D372C 1 & 2 25.31 9.14E−10 0.49 E103A 1 & 2 26.74 9.00E−10 0.48 K127H 1 & 2 7.84 8.91E−10 0.48 Q323A 1 & 2 32.81 8.75E−10 0.47 Q119F 1 & 2 7.96 8.68E−10 0.47 Q291G 1 & 2 24.16 8.65E−10 0.47 L82I 1 & 2 18.72 8.57E−10 0.46 H391K 1 & 2 39.76 8.27E−10 0.44 M414T 1 & 2 19.07 8.25E−10 0.44 N392H 1 & 2 18.32 8.16E−10 0.44 K127R 1 & 2 19.72 8.09E−10 0.44 S76D 1 & 2 25.46 8.04E−10 0.43 Q119E 1 & 2 3.76 8.02E−10 0.43 K127P 1 & 2 15.16 8.01E−10 0.43 Q323R 1 & 2 44.35 7.73E−10 0.42 K86S 1 & 2 22.38 7.65E−10 0.41 M414C 1 & 2 22.85 7.56E−10 0.41 K63S 1 & 2 25.74 7.44E−10 0.40 A57V 1 & 2 26.82 7.31E−10 0.39 E224G 1 & 2 19.68 7.28E−10 0.39 K127M 1 & 2 25.96 7.08E−10 0.38 S387Q 1 & 2 13.81 6.91E−10 0.37 Q119K 1 & 2 7.03 6.84E−10 0.37 E436Q 1 & 2 13.28 6.66E−10 0.36 K127L 1 & 2 16.56 6.36E−10 0.34 K127Q 1 & 2 13.44 6.27E−10 0.34 M414E 1 & 2 29.35 6.23E−10 0.33 Q323L 1 & 2 21.44 6.19E−10 0.33 K127A 1 & 2 8.08 6.14E−10 0.33 Q159K 1 & 2 66.10 5.94E−10 0.32 K127V 1 & 2 32.84 5.12E−10 0.28 K127G 1 & 2 6.92 5.09E−10 0.27 K127Y 1 & 2 14.04 4.98E−10 0.27 K127S 1 & 2 5.64 4.97E−10 0.27 K127D 1 & 2 80.64 4.91E−10 0.26 Q119L, Q323H 1 & 2 114.45 4.82E−10 0.26 Y129W 1 & 2 270.88 4.45E−10 0.24 K127I 1 & 2 31.28 4.42E−10 0.24 K127W 1 & 2 9.16 4.40E−10 0.24 V106A 1 & 2 26.68 4.28E−10 0.23 K127T 1 & 2 21.00 3.54E−10 0.19 K127C 1 & 2 10.76 3.42E−10 0.18 K127E 1 & 2 6.60 1.63E−10 0.09

Example 5 Engineering PPO-Derivative Herbicide Tolerant Plants having Wildtype or Mutated PPO Sequences

PPO-derivative herbicide tolerant soybean (Glyceine max, corn (Zea mays), and Canola (Brassica napus or Brassica Rapa var. or Brassica campestris L) plants are produced by a method as described by Olhoft et al. (US patent 2009/0049567). For transformation of soybean or Arabidopsis thaliana, Wildtype or Mutated PPO sequences encoding mutated PPO polypeptides comprising SEQ ID NO: 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, 100, 101, 102, 103, 104, 105, 106, 107, 108, 109, 110, 111, 112, 113, 114, 115, 116, 117, or 129, are cloned with standard cloning techniques as described in Sambrook et al. (Molecular cloning (2001) Cold Spring Harbor Laboratory Press) in a binary vector containing resistance marker gene cassette (AHAS) and mutated PPO sequence (marked as GOI) in between ubiquitin promoter (PcUbi) and nopaline synthase terminator (NOS) sequence. For corn transformation, Wildtype or Mutated PPO sequences are cloned with standard cloning techniques as described in Sambrook et al. (Molecular cloning (2001) Cold Spring Harbor Laboratory Press) in a binary vector containing resistance marker gene cassette (AHAS) and mutated PPO sequence (marked as GOI) in between corn ubiquitin promoter (ZmUbi) and nopaline synthase terminator (NOS) sequence. Binary plasmids are introduced to Agrobacterium tumefaciens for plant transformation. Plasmid constructs are introduced into soybean's axillary meristem cells at the primary node of seedling explants via Agrobacterium-mediated transformation. After inoculation and co-cultivation with Agrobacteria, the explants are transferred to shoot introduction media without selection for one week. The explants are subsequently transferred to a shoot induction medium with 1-3 μM imazapyr (Arsenal) for 3 weeks to select for transformed cells. Explants with healthy callus/shoot pads at the primary node are then transferred to shoot elongation medium containing 1-3 μM imazapyr until a shoot elongated or the explant died. Transgenic plantlets are rooted, subjected to TaqMan analysis for the presence of the transgene, transferred to soil and grown to maturity in greenhouse. Transformation of corn plants are done by a method described by McElver and Singh (WO 2008/124495). Plant transformation vector constructs containing mutated PPO sequences are introduced into maize immature embryos via Agrobacterium-mediated transformation.

Transformed cells are selected in selection media supplemented with 0.5-1.5 μM imazethapyr for 3-4 weeks. Transgenic plantlets are regenerated on plant regeneration media and rooted afterwards. Transgenic plantlets are subjected to TaqMan analysis for the presence of the transgene before being transplanted to potting mixture and grown to maturity in greenhouse. Arabidopsis thaliana are transformed with wildtype or mutated PPO sequences by floral dip method as described by McElver and Singh (WO 2008/124495). Transgenic Arabidopsis plants are subjected to TaqMan analysis for analysis of the number of integration loci. Transformation of Oryza sativa (rice) are done by protoplast transformation as described by Peng et al. (U.S. Pat. No. 6,653,529) T0 or T1 transgenic plant of soybean, corn, and rice containing mutated PPO sequences are tested for improved tolerance to PPO-derived herbicides in greenhouse studies and mini-plot studies with the following PPO-inhibiting herbicides: saflufenacil, 1,5-dimethyl-6-thioxo-3-(2,2,7-trifluoro-3-oxo-4-(prop-2-ynyl)-3,4-dihydro-2H-benzo[b][1,4]oxazin-6-yl)-1,3,5-triazinane-2,4-dione (CAS 1258836-72-4/trifludimoxazin), flumioxazin, butafenacil, acifluorfen, lactofen, bifenox, sulfentrazone, and photosynthesis inhibitor diuron as negative control.

Transgenic Arabidopsis thaliana plants are assayed for improved tolerance to saflufenacil, 1,5-dimethyl-6-thioxo-3-(2,2,7-trifluoro-3-oxo-4-(prop-2-ynyl)-3,4-dihydro-2H-benzo[b][1,4]oxazin-6-yl)-1,3,5-triazinane-2,4-dione (CAS 1258836-72-4/trifludimoxazin), flumioxazin, butafenacil, acifluorfen, lactofen, bifenox, sulfentrazone, and photosynthesis inhibitor diuron as negative control in 48-well plates. Therefore, T2 seeds are surface sterilized by stirring for 5 min in ethanol+water (70+30 by volume), rinsing one time with ethanol+water (70+30 by volume) and two times with sterile, deionized water. The seeds are resuspended in 0.1% agar dissolved in water (w/v) Four to five seeds per well are plated on solid nutrient medium consisting of half-strength murashige skoog nutrient solution, pH 5.8 (Murashige and Skoog (1962) Physiologia Plantarum 15: 473-497). Compounds are dissolved in dimethylsulfoxid (DMSO) and added to the medium prior solidification (final DMSO concentration 0.1%). Multi well plates are incubated in a growth chamber at 22° C., 75% relative humidity and 110 μmol Phot*m⁻²*s⁻¹ with 14:10 h light:dark photoperiod. Growth inhibition is evaluated seven to ten days after seeding in comparison to wild type plants.

Additionally, transgenic T1 Arabidopsis plants are tested for improved tolerance to PPO-inhibiting herbicides in greenhouse studies with the following PPO-inhibiting herbicides: saflufenacil, 1,5-dimethyl-6-thioxo-3-(2,2,7-trifluoro-3-oxo-4-(prop-2-ynyl)-3,4-dihydro-2H-benzo[b][1,4]oxazin-6-yl)-1,3,5-triazinane-2,4-dione (CAS 1258836-72-4/trifludimoxazin), flumioxazin, butafenacil, acifluorfen, lactofen, bifenox, sulfentrazone, and photosynthesis inhibitor diuron as negative control.

Additionally, transgenic T2 Arabidopsis plants are tested for improved tolerance to PPO-inhibiting herbicides. Plants are treated with the following herbicides in the greenhouse: Saflufenacil, 1,5-dimethyl-6-thioxo-3-(2,2,7-trifluoro-3-oxo-4-(prop-2-ynyl)-3,4-dihydro-2H-benzo[b][1,4]oxazin-6-yl)-1,3,5-triazinane-2,4-dione (CAS 1258836-72-4/trifludimoxazin), Uracilpyridine 2, Uracilpyridine 4, Uracilpyridine 1, Sulfentrazone, and Flumioxazin+1% MSO. Results are shown in FIGS. 3 through 11.

Example 6 Tissue Culture Conditions

An in vitro tissue culture mutagenesis assay has been developed to isolate and characterize plant tissue (e.g., maize, rice tissue) that is tolerant to protoporphyrinogen oxidase inhibiting herbicides, (saflufenacil, 1,5-dimethyl-6-thioxo-3-(2,2,7-trifluoro-3-oxo-4-(prop-2-ynyl)-3,4-dihydro-2H-benzo[b][1,4]oxazin-6-yl)-1,3,5-triazinane-2,4-dione (CAS 1258836-72-4/trifludimoxazin), flumioxazin, butafenacil, acifluorfen, lactofen, bifenox, sulfentrazone, and photosynthesis inhibitor diuron as negative control). The assay utilizes the somaclonal variation that is found in in vitro tissue culture. Spontaneous mutations derived from somaclonal variation can be enhanced by chemical mutagenesis and subsequent selection in a stepwise manner, on increasing concentrations of herbicide.

The present invention provides tissue culture conditions for encouraging growth of friable, embryogenic maize or rice callus that is regenerable. Calli are initiated from 4 different maize or rice cultivars encompassing Zea mays and Japonica (Taipei 309, Nipponbare, Koshihikari) and Indica (Indica 1) varieties, respectively. Seeds are surface sterilized in 70% ethanol for approximately 1 min followed by 20% commercial Clorox bleach for 20 minutes. Seeds are rinsed with sterile water and plated on callus induction media. Various callus induction media are tested. The ingredient lists for the media tested are presented in Table Y.

TABLE Y Ingredient Supplier R001M R025M R026M R327M R008M MS711R B5 Vitamins Sigma 1.0 X MS salts Sigma 1.0 X 1.0 X 1.0 X 1.0 X MS Vitamins Sigma 1.0 X 1.0 X N6 salts Phytotech 4.0 g/L 4.0 g/L N6 vitamins Phytotech 1.0 X 1.0 X L-Proline Sigma 2.9 g/L 0.5 g/L 1.2 g/L Casannino BD 0.3 g/L 0.3 g/L 2 g/L Acids Casein Sigma 1.0 g/L Hydrolysate L-Asp Phytotech 150 mg/L Monohydrate Nicotinic Acid Sigma 0.5 mg/L Pyridoxine HCl Sigma 0.5 mg/L Thiamine HCl Sigma 1.0 mg/L Myo-inositol Sigma 100 mg/L MES Sigma 500 mg/L 500 mg/L 500 mg/L 500 mg/L 500 mg/L 500 mg/L Maltose VWR 30 g/L 30 g/L 30 g/L 30 g/L Sorbitol Duchefa 30 g/L Sucrose VWR 10 g/L 30 g/L NAA Duchefa 50 μg/L 2,4-D Sigma 2.0 mg/L 1.0 mg/L MgC12.6H20 VWR 750 mg/L →pH 5.8 5.8 5.8 5.8 5.8 5.7 Gelrite Duchefa 4.0 g/L 2.5 g/L Agarose Type1 Sigma 7.0 g/L 10 g/L 10 g/L →Autoclave 15 min 15 min 15 min 15 min 15 min 20 min Kinetin Sigma 2.0 mg/L 2.0 mg/L NAA Duchefa 1.0 mg/L 1.0 mg/L ABA Sigma 5.0 mg/L Cefotaxinne Duchefa 0.1 g/L 0.1 g/L 0.1 g/L Vancomycin Duchefa 0.1 g/L 0.1 g/L 0.1 g/L G418 Disulfate Sigma 20 mg/L 20 mg/L 20 mg/L

R001M callus induction media is selected after testing numerous variations. Cultures are kept in the dark at 30° C. Embryogenic callus is subcultured to fresh media after 10-14 days.

Example 7 Selection of Herbicide-Tolerant Calli

Once tissue culture conditions are determined, further establishment of selection conditions are established through the analysis of tissue survival in kill curves with saflufenacil, 1,5-dimethyl-6-thioxo-3-(2,2,7-trifluoro-3-oxo-4-(prop-2-ynyl)-3,4-dihydro-2H-benzo[b][1,4]oxazin-6-yl)-1,3,5-triazinane-2,4-dione (CAS 1258836-72-4/trifludimoxazin), flumioxazin, butafenacil, acifluorfen, lactofen, bifenox, sulfentrazone, and photosynthesis inhibitor diuron as negative control. Careful consideration of accumulation of the herbicide in the tissue, as well as its persistence and stability in the cells and the culture media is performed. Through these experiments, a sub-lethal dose has been established for the initial selection of mutated material. After the establishment of the starting dose of saflufenacil, 1,5-dimethyl-6-thioxo-3-(2,2,7-trifluoro-3-oxo-4-(prop-2-ynyl)-3,4-dihydro-2H-benzo[b][1,4]oxazin-6-yl)-1,3,5-triazinane-2,4-dione (CAS 1258836-72-4/trifludimoxazin), flumioxazin, butafenacil, acifluorfen, lactofen, bifenox, sulfentrazone, and photosynthesis inhibitor diuron as negative control in selection media, the tissues are selected in a step-wise fashion by increasing the concentration of the PPO inhibitor with each transfer until cells are recovered that grew vigorously in the presence of toxic doses. The resulting calli are further subcultured every 3-4 weeks to R001M with selective agent. Over 26,000 calli are subjected to selection for 4-5 subcultures until the selective pressure is above toxic levels as determined by kill curves and observations of continued culture. Alternatively, liquid cultures initiated from calli in MS711R with slow shaking and weekly subcultures. Once liquid cultures are established, selection agent is added directly to the flask at each subculture. Following 2-4 rounds of liquid selection, cultures are transferred to filters on solid R001M media for further growth.

Example 8 Regeneration of Plants

Tolerant tissue is regenerated and characterized molecularly for PPO gene sequence mutations and/or biochemically for altered PPO activity in the presence of the selective agent. In addition, genes involved directly and/or indirectly in tetrapyrrole biosynthesis and/or metabolism pathways are also sequenced to characterize mutations. Finally, enzymes that change the fate (e.g. metabolism, translocation, transportation) are also sequence to characterized mutations. Following herbicide selection, calli are regenerated using a media regime of R025M for 10-14 days, R026M for ca. 2 weeks, R327M until well formed shoots are developed, and R0085 until shoots are well rooted for transfer to the greenhouse. Regeneration is carried out in the light. No selection agent is included during regeneration. Once strong roots are established, M0 regenerants are transplant to the greenhouse in square or round pots. Transplants are maintained under a clear plastic cup until they are adapted to greenhouse conditions. The greenhouse is set to a day/night cycle of 27° C./21° C. (80° F./70° F.) with 600 W high pressure sodium lights supplementing light to maintain a 14-hour day length. Plants are watered according to need, depending in the weather and fertilized daily.

Example 9 Sequence Analysis

Leaf tissue is collected from clonal plants separated for transplanting and analyzed as individuals. Genomic DNA is extracted using a Wizard® 96 Magnetic DNA Plant System kit (Promega, U.S. Pat. Nos. 6,027,945 & 6,368,800) as directed by the manufacturer. Isolated DNA is PCR amplified using the appropriate forward and reverse primer.

PCR amplification is performed using Hotstar Taq DNA Polymerase (Qiagen) using touchdown thermocycling program as follows: 96° C. for 15 min, followed by 35 cycles (96° C., 30 sec; 58° C.-0.2° C. per cycle, 30 sec; 72° C., 3 min and 30 sec), 10 min at 72° C. PCR products are verified for concentration and fragment size via agarose gel electrophoresis. Dephosphorylated PCR products are analyzed by direct sequence using the PCR primers (DNA Landmarks, or Entelechon). Chromatogram trace files (.scf) are analyzed for mutation relative to the wild-type gene using Vector NTI Advance 10™ (Invitrogen). Based on sequence information, mutations are identified in several individuals. Sequence analysis is performed on the representative chromatograms and corresponding AlignX alignment with default settings and edited to call secondary peaks.

Example 10 Demonstration of Herbicide-Tolerance

T0 or T1 transgenic plant of soybean, corn, Canola varieties and rice containing PPO1 and or PPO2 sequences are tested for improved tolerance to herbicides in greenhouse studies and mini-plot studies with the following herbicides: saflufenacil, 1,5-dimethyl-6-thioxo-3-(2,2,7-trifluoro-3-oxo-4-(prop-2-ynyl)-3,4-dihydro-2H-benzo[b][1,4]oxazin-6-yl)-1,3,5-triazinane-2,4-dione (CAS 1258836-72-4/trifludimoxazin), flumioxazin, butafenacil, acifluorfen, lactofen, bifenox, sulfentrazone, and photosynthesis inhibitor diuron as negative control. For the pre-emergence treatment, the herbicides are applied directly after sowing by means of finely distributing nozzles. The containers are irrigated gently to promote germination and growth and subsequently covered with transparent plastic hoods until the plants have rooted. This cover causes uniform germination of the test plants, unless this has been impaired by the herbicides. For post emergence treatment, the test plants are first grown to a height of 3 to 15 cm, depending on the plant habit, and only then treated with the herbicides. For this purpose, the test plants are either sown directly, and grown in the same containers or they are first grown separately and transplanted into the test containers a few days prior to treatment.

For testing of TO plants, cuttings can be used. In the case of soybean plants, an optimal shoot for cutting is about 7.5 to 10 cm tall, with at least two nodes present. Each cutting is taken from the original transformant (mother plant) and dipped into rooting hormone powder (indole-3-butyric acid, IBA). The cutting is then placed in oasis wedges inside a bio-dome. Wild type cuttings are also taken simultaneously to serve as controls. The cuttings are kept in the bio-dome for 5-7 days and then transplanted to pots and then acclimated in the growth chamber for two more days. Subsequently, the cuttings are transferred to the greenhouse, acclimated for approximately 4 days, and then subjected to spray tests as indicated. Depending on the species, the plants are kept at 10-25° C. or 20-35° C. The test period extends over 3 weeks. During this time, the plants are tended and their response to the individual treatments is evaluated. Herbicide injury evaluations are taken at 2 and 3 weeks after treatment. Plant injury is rated on a scale of 0% to 100%, 0% being no injury and 100% being complete death.

Transgenic Arabidopsis thaliana plants are assayed for improved tolerance to saflufenacil, 1,5-dimethyl-6-thioxo-3-(2,2,7-trifluoro-3-oxo-4-(prop-2-ynyl)-3,4-dihydro-2H-benzo[b][1,4]oxazin-6-yl)-1,3,5-triazinane-2,4-dione (CAS 1258836-72-4/trifludimoxazin), flumioxazin, butafenacil, acifluorfen, lactofen, bifenox, sulfentrazone, and photosynthesis inhibitor diuron as negative control, in 48-well plates. Therefore, T2 seeds are surface sterilized by stirring for 5 min in ethanol+water (70+30 by volume), rinsing one time with ethanol+water (70+30 by volume) and two times with sterile, deionized water. The seeds are resuspended in 0.1% agar dissolved in water (w/v) Four to five seeds per well are plated on solid nutrient medium consisting of half-strength murashige skoog nutrient solution, pH 5.8 (Murashige and Skoog (1962) Physiologia Plantarum 15: 473-497). Compounds are dissolved in dimethylsulfoxid (DMSO) and added to the medium prior solidification (final DMSO concentration 0.1%). Multi well plates are incubated in a growth chamber at 22° C., 75% relative humidity and 110 μmol Phot*m⁻²*s⁻¹ with 14:10 h light:dark photoperiod. Growth inhibition is evaluated seven to ten days after seeding in comparison to wild type plants. Additionally, transgenic T1 Arabidopsis plants are tested for improved tolerance to herbicides in greenhouse studies with the following herbicides: saflufenacil, 1,5-dimethyl-6-thioxo-3-(2,2,7-trifluoro-3-oxo-4-(prop-2-ynyl)-3,4-dihydro-2H-benzo[b][1,4]oxazin-6-yl)-1,3,5-triazinane-2,4-dione (CAS 1258836-72-4/trifludimoxazin), flumioxazin, butafenacil, acifluorfen, lactofen, bifenox, sulfentrazone, and photosynthesis inhibitor diuron as negative control. The results are shown in the following table and in FIGS. 1 and 2.

Germination Assay - trangenic ARBTH germinating in medium containing selection agent Tolerance Factor Event Species_gene_substitution Compound IC 50 (M) (non transgeneic = 1.0) non-transgenic (MC-24) Saflufenacil 2.50E−08 1 AMATU_PPO2_wild_type Saflufenacil 1.06E−07 10 D AMATU_PPO2_G211A_F420M Saflufenacil 2.50E−05 1000 M AMATU_PPO2_G211A_F420M Saflufenacil 2.50E−05 1000 P AMATU_PPO2_G211A_F420M Saflufenacil 7.50E−06 300 Q AMATU_PPO2_G211A_F420M Saflufenacil 7.50E−06 300 R AMATU_PPO2_G211A_F420M Saflufenacil 7.50E−06 300

Example 11 Herbicide Selection Using Tissue Culture

Media is selected for use and kill curves developed as specified above. For selection, different techniques are utilized. Either a step wise selection is applied, or an immediate lethal level of herbicide is applied. In either case, all of the calli are transferred for each new round of selection. Selection is 4-5 cycles of culture with 3-5 weeks for each cycle. Cali are placed onto nylon membranes to facilitate transfer (200 micron pore sheets, Biodesign, Saco, Maine). Membranes are cut to fit 100×20 mm Petri dishes and are autoclaved prior to use 25-35 calli (average weight/calli being 22 mg) are utilized in every plate. In addition, one set of calli are subjected to selection in liquid culture media with weekly subcultures followed by further selection on semi-solid media. Mutant lines are selected using saflufenacil, 1,5-dimethyl-6-thioxo-3-(2,2,7-trifluoro-3-oxo-4-(prop-2-ynyl)-3,4-dihydro-2H-benzo[b][1,4]oxazin-6-yl)-1,3,5-triazinane-2,4-dione (CAS 1258836-72-4/trifludimoxazin), flumioxazin, butafenacil, acifluorfen, lactofen, bifenox, sulfentrazone, and photosynthesis inhibitor diuron as negative control. Efficiencies of obtaining mutants is high either based on a percentage of calli that gave rise to a regenerable, mutant line or the number of lines as determined by the gram of tissue utilized.

Example 12 Maize Whole Plant Transformation and PPO Inhibitor Tolerance Testing

Immature embryos are transformed according to the procedure outlined in Peng et al. (WO2006/136596). Plants are tested for the presence of the T-DNA by Taqman analysis with the target being the nos terminator which is present in all constructs. Healthy looking plants are sent to the greenhouse for hardening and subsequent spray testing. The plants are individually transplanted into MetroMix 360 soil in 4″ pots. Once in the greenhouse (day/night cycle of 27° C./21° C. with 14 hour day length supported by 600 W high pressure sodium lights), they are allowed to grow for 14 days. They are then sprayed with a treatment of 25 to 200 g ai/ha saflufenacil+1.0% v/v methylated seed oil (MSO) and/or 25-200 g ai/ha 1,5-dimethyl-6-thioxo-3-(2,2,7-trifluoro-3-oxo-4-(prop-2-ynyl)-3,4-dihydro-2H-benzo[b][1,4]oxazin-6-yl)-1,3,5-triazinane-2,4-dione (CAS 1258836-72-4/trifludimoxazin) plus 1% MSO. Other PPO inhibiting herbicides are also tested in a similar fashion for confirming cross resistance: flumioxazin, butafenacil, acifluorfen, lactofen, bifenox, sulfentrazone, and photosynthesis inhibitor diuron as negative control. Herbicide injury evaluations are taken at 7, 14 and 21 days after treatment. Herbicide injury evaluations are taken 2, 7, 14 and 21 days post-spray to look for injury to new growth points and overall plant health. The top survivors are transplanted into gallon pots filled with MetroMix 360 for seed production.

In addition, T1 transgenic maize harboring Amtu_PPO2 variants are tested for tolerance for pre-emergence application of Saflufenacil. Seeds are planted in a mixture of sand an Metromix and then immediately treated with 50 and 200 g ai/ha Saflufenacil. Seeds and plantlets are assessed for germination and root length. Results are shown in FIG. 14.

Example 13 Soybean Transformation and PPO Inhibitor Tolerance Testing

Soybean cv Jake is transformed as previously described by Siminszky et al., Phytochem Rev. 5:445-458 (2006). After regeneration, transformants are transplanted to soil in small pots, placed in growth chambers (16 hr day/8 hr night; 25° C. day/23° C. night; 65% relative humidity; 130-150 microE m−2 s−1) and subsequently tested for the presence of the T-DNA via Taqman analysis. After a few weeks, healthy, transgenic positive, single copy events are transplanted to larger pots and allowed to grow in the growth chamber. An optimal shoot for cutting is about 3-4 inches tall, with at least two nodes present. Each cutting is taken from the original transformant (mother plant) and dipped into rooting hormone powder (indole-3-butyric acid, IBA). The cutting is then placed in oasis wedges inside a bio-dome. The mother plant is taken to maturity in the greenhouse and harvested for seed. Wild type cuttings are also taken simultaneously to serve as negative controls. The cuttings are kept in the bio-dome for 5-7 days and then transplanted to 3 inch pots and then acclimated in the growth chamber for two more days. Subsequently, the cuttings are transferred to the greenhouse, acclimated for approximately 4 days, and then sprayed with a treatment of 0-200 g ai/ha saflufenacil plus 1% MSO and/or 25-200 g ai/ha 1,5-dimethyl-6-thioxo-3-(2,2,7-trifluoro-3-oxo-4-(prop-2-ynyl)-3,4-dihydro-2H-benzo[b][1,4]oxazin-6-yl)-1,3,5-triazinane-2,4-dione (CAS 1258836-72-4/trifludimoxazin) plus 1% MSO. Other PPO inhibiting herbicides are also tested in a similar fashion for confirming cross resistance: flumioxazin, butafenacil, acifluorfen, lactofen, bifenox, sulfentrazone, and photosynthesis inhibitor diuron as negative control. Herbicide injury evaluations are taken at 7 days after treatment.

Results are shown in FIGS. 12 and 13.

The following gives a definition of the injury scores measured above:

ScoreDescription of injury

0 No Injury

1 Minimal injury, only a few patches of leaf injury or chlorosis.

2 Minimal injury with slightly stronger chlorosis. Overall growth points remain undamaged.

3 Slightly stronger injury on secondary leaf tissue, but primary leaf and growth points are still undamaged.

4 Overall plant morphology is slightly different, some chlorosis and necrosis in secondary growth points and leaf tissue. Stems are intact. Regrowth is highly probable within 1 week.

5 Overall plant morphology is clearly different, some chlorosis and necrosis on a few leaves and growth points, but primary growth point is intact. Stem tissue is still green. Regrowth is highly probably within 1 week.

6 Strong injury can be seen on the new leaflet growth. Plant has a high probability to survive only through regrowth at different growth points. Most of the leaves are chlorotic/necrotic but stem tissue is still green. May have regrowth but with noticeable injured appearance.

7 Most of the active growth points are necrotic. There may be a single growth point that could survive and may be partially chlorotic or green and partially necrotic. Two leaves may still be chlorotic with some green; the rest of the plant including stem is necrotic.

8 Plant will likely die, and all growth points are necrotic. One leaf may still be chlorotic with some green. The remainder of the plant is necrotic.

9 Plant is dead.

* Not tested

Example 14 Transient Protein Expression in Tobacco Leafs

Transient expression of mutated PPO sequences as wildtype or with respective mutations were done as described previously (Voinnet O., et al., 2003, The Plant Journal 33, 949-956). In brief, cloning of GOI and Agrobacterium transformation (strain: GV2260) were done as described in EXAMPLE 5. Young leaves of Nicotiana benthamiana were infiltrated with transgenic Agrobacterium suspension (OD⁶⁰⁰ of 1.0) harboring binary vector constructs containing a GOI gene controlled by a promoter and terminator sequence. 48 h to 72 h after infiltration punches of leave discs (0.75 cm in diameter) were transferred to 6-well plates with medium (half strength Linsmaier-Skoog (Linsmaier and Skoog (1965) Physiol. Plant. 18: 100-127) nutrient solution or water) containing herbicide of interest in different concentrations. Multi well plates were incubated in a growth chamber at 22° C., 75% relative humidity and 110 μmol Phot*m⁻²*s⁻¹ with 14:10 h light: dark photoperiod.

Example 15 Demonstration of Herbicide Tolerance of Transiently Transformed Tobacco Leaf Discs

Leaf discs, generated as described in EXAMPLE 14, expressing a protein encoded by GOI, were subjected to analysis on improved tolerance to herbicide treatment. For analysis of herbicide damage, chlorophyll fluorescence were identified as indicative marker (Dayan and Zaccaro (2012) Pest. Biochem. Physiol. 102: 189-197). In addition to monitor herbicide effect by visual inspection the photosynthetic yield of photosystem II were done with a MAXI imaging PAM machine (IMAGINE-PAM M-Series, Walz, Effeltrich, Germany) 48 h after starting herbicide treatment. PSII yield were measured as per manufacturer instructions. Tolerance factors were calculated based on IC₅₀ values of PSII yield inhibition of transformed versus empty vector-transformed leaf discs. IC₅₀ of PSII yield inhibition in empty vector-transformed leaf discs treated with Saflufenacil or 1,5-dimethyl-6-thioxo-3-(2,2,7-trifluoro-3-oxo-4-(prop-2-ynyl)-3,4-dihydro-2H-benzo[b][1,4]oxazin-6-yl)-1,3,5-triazinane-2,4-dione for 48 h was measured with 1.1*10⁻⁷ M or 1.1*10⁻⁸ M, respectively. 

1. A plant or plant part comprising a polynucleotide encoding a mutated PPO polypeptide, the expression of said polynucleotide confers to the plant or plant part tolerance to herbicides, wherein said mutated PPO polypeptide comprises one or more of the following motifs. 1) Motif 1: GT[C/S]GGDP (SEQ ID NO: 131) Wherein the glycine at position 4, and/or 5 within said motif of the corresponding wildtype sequence is substituted by any other amino acid 2) Motif 2: [A/S/C]PS[D/N][X][X]L (SEQ ID NO: 132) Wherein the serine at position 3 within said motif of the corresponding wildtype sequence is substituted by any other amino acid 3) Motif 3: [R/Q][E/D]KQQ[L/Y]P (SEQ ID NO: 133) Wherein the glutamine at position 4 within said motif of the corresponding wildtype sequence is substituted by any other amino acid 4) Motif 4: L[I/V]PSKE (SEQ ID NO: 134) wherein the serine at position 4 within said motif of the corresponding wildtype sequence is substituted by any other amino acid.
 2. The plant or plant part of claim 1, wherein the mutated PPO polypeptide in addition comprises one or more of the following motifs a. Motif 5: SQ[N/K/H]KRYI (SEQ IS NO:135), wherein the Arg at position 5 within said motif is substituted by any other amino acid; b. Motif 6: TLGTLFSS (SEQ ID NO: 136), wherein the Leu at position 2 within said motif is substituted by any other amino acid; c. Motif 7: [F/Y]TTF[V/I]GG (SEQ ID NO: 137), wherein the Phe at position 4 within said motif is substituted by any other amino acid.
 3. The plant or plant part of claim 1, wherein said mutated PPO polypeptide comprises a combination of Motifs, said combination is selected from the group consisting of: Motif 5—Motif 1—Motif-2—Motif 7; Motif 5—Motif 1—Motif 7, Motif 5—Motif 4—Motif 7; Motif 3—Motif 5—Motif 7; Motif 1—Motif 7; and Motif 1—Motif 6—Motif
 7. 4. The plant or plant part of claim 1, wherein the polynucleotide encoding the mutated PPO polypeptide comprises the nucleic acid sequence set forth in SEQ ID NO: 118, 119, 120, 121, 122, 123, 124, 125, 126, 127, 128 or 130, or a homologue, variant or derivative thereof.
 5. The plant or plant part of claim 1, wherein the mutated PPO polypeptide is a functional variant having, over the full-length of the variant, at least about 60%, illustratively, at least about 80%, 90%, 95%, 98%, 99% or more amino acid sequence identity to SEQ ID NO: 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, 100, 101, 102, 103, 104, 105, 106, 107, 108, 109, 110, 111, 112, 113, 114, 115, 116, 117 or
 129. 6. The plant or plant part of claim 1, wherein the mutated PPO refers to a PPO polypeptide the amino acid sequence of which differs from the respective wildtype amino acid sequence at one or more positions corresponding to positions 32, 53, 57, 61, 63, 64, 65, 67, 71, 76, 82, 83, 85, 86, 87, 88, 91, 103, 104, 106, 108, 116, 119, 126, 127, 129, 139, 159, 210, 211, 224, 245, 246, 248, 249, 252, 253, 254, 255, 257, 259, 260, 262, 264, 286, 291, 305, 308, 309, 323, 335, 343, 345, 358, 372, 373, 387, 391, 392, 400, 412, 414, 415, 425, 428, 431, 433, 434, 435, 436, 447, 451, 453, 464, 466, 482 of SEQ ID NO: 1 or 2, wherein said difference refers to a substitution of the amino acid at that positions by any other amino acid.
 7. A seed capable of germination into a plant comprising in at least some of its cells a polynucleotide operably linked to a promoter operable in plant cells, the promoter capable of expressing a mutated PPO polypeptide encoded by the polynucleotide as defined in claim 1, the expression of the mutated PPO polypeptide conferring to the plant tolerance to herbicides.
 8. A plant cell of or capable of regenerating a plant comprising in at least some of its cells a polynucleotide operably linked to a promoter operable in plant cells, the promoter capable of expressing a mutated PPO polypeptide encoded by the polynucleotide as defined in claim 1, the expression of the mutated PPO polypeptide conferring to the plant tolerance to herbicides, wherein the plant cell comprises the polynucleotide operably linked to a promoter.
 9. A plant cell comprising a polynucleotide operably linked to a promoter operable in a cell, the promoter capable of expressing a mutated PPO polypeptide encoded by the polynucleotide as defined in claim 1, the expression of the mutated PPO polypeptide conferring to the plant tolerance to herbicides.
 10. A plant product prepared from a plant or plant part comprising in at least some of its cells a polynucleotide operably linked to a promoter operable in plant cells, the promoter capable of expressing a mutated PPO polypeptide encoded by the polynucleotide as defined in claim 1, the expression of the mutated PPO polypeptide conferring to the plant tolerance to herbicides.
 11. A progeny or descendant plant derived from a plant comprising in at least some of its cells a polynucleotide operably linked to a promoter operable in plant cells, the promoter capable of expressing a mutated PPO polypeptide encoded by the polynucleotide as defined in claim 1, wherein the progeny or descendant plant comprises in at least some of its cells the polynucleotide operably linked to the promoter, the expression of the mutated PPO polypeptide conferring to the progeny or descendant plant tolerance to the herbicides.
 12. A method for controlling weeds at a locus for growth of a plant, the method comprising: (a) applying an herbicide composition comprising herbicides to the locus; and (b) planting a seed at the locus, wherein the seed is capable of producing a plant that comprises in at least some of its cells a polynucleotide operably linked to a promoter operable in plant cells, the promoter capable of expressing a mutated PPO polypeptide encoded by the polynucleotide as defined in claim 1, the expression of the mutated PPO polypeptide conferring to the plant tolerance to herbicides.
 13. The method of claim 12, wherein herbicide composition is applied to the weeds and to the plant produced by the seed.
 14. A method of producing a plant having tolerance to herbicide, the method comprising regenerating a plant from a plant cell transformed with a recombinant polynucleotide operably linked to a promoter operable in plant cells, the promoter capable of expressing a mutated PPO polypeptide encoded by the polynucleotide as defined in claim 1, the expression of the mutated PPO polypeptide conferring to the plant tolerance to herbicides.
 15. A method of producing a progeny plant having tolerance to herbicide, the method comprising: crossing a first herbicide-tolerant plant with a second plant to produce a herbicide-tolerant progeny plant, wherein the first plant and the progeny plant comprise in at least some of their cells a polynucleotide operably linked to a promoter operable in plant cells, the promoter capable of expressing a mutated PPO polypeptide encoded by the polynucleotide as defined in claim 1, the expression of the mutated PPO polypeptide conferring to the plant tolerance to herbicides.
 16. A plant or plant part comprising in at least some of its cells a polynucleotide operably linked to a promoter operable in plant cells, the promoter capable of expressing a mutated PPO polypeptide encoded by the polynucleotide as defined in claim 1, the expression of the mutated PPO polypeptide conferring to the plant tolerance to herbicides, wherein the plant or plant part further exhibits a second or third herbicide-tolerant trait.
 17. An isolated and/or recombinantly produced and/or synthetic nucleic acid molecule comprising a nucleic acid molecule encoding a mutated PPO polypeptide selected from the group consisting of: (a) a nucleic acid molecule encoding a mutated PPO polypeptide comprising the sequence of SEQ ID NO: 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, 100, 101, 102, 103, 104, 105, 106, 107, 108, 109, 110, 111, 112, 113, 114, 115, 116, 117 or 129, or a variant, paralogue, orthologue or homolog thereof; (b) a nucleic acid molecule, which, as a result of the degeneracy of the genetic code, can be derived from a nucleic acid molecule encoding a mutated PPO polypeptide comprising the sequence of SEQ ID NO: 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, 100, 101, 102, 103, 104, 105, 106, 107, 108, 109, 110, 111, 112, 113, 114, 115, 116, 117 or 129, or a variant, paralogue, orthologue or homolog thereof, and confers increased herbicide tolerance or resistance, as compared to a corresponding, non-transformed, wild type plant cell, a plant or a part thereof; (c) a nucleic acid molecule encoding a mutated PPO polypeptide having 30% or more identity with the amino acid sequence of the PPO polypeptide sequence of SEQ ID NO: 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, 100, 101, 102, 103, 104, 105, 106, 107, 108, 109, 110, 111, 112, 113, 114, 115, 116, 117 or 129, and confers increased herbicide tolerance or resistance, as compared to a corresponding, non-transformed, wild type plant cell, a plant or a part thereof; (d) nucleic acid molecule which hybridizes with a nucleic acid molecule of (a), (b), or (c), under stringent hybridization conditions and confers increased herbicide tolerance or resistance, as compared to a corresponding, non-transformed, wild type plant cell, a plant or a part thereof; wherein the amino acid sequence of the encoded mutated PPO polypeptide differs from the wildtype amino acid sequence of a PPO polypeptide at one or more positions corresponding to positions 32, 53, 57, 61, 63, 64, 65, 67, 71, 76, 82, 83, 85, 86, 87, 88, 91, 103, 104, 106, 108, 116, 119, 126, 127, 129, 139, 159, 210, 211, 224, 245, 246, 248, 249, 252, 253, 254, 255, 257, 259, 260, 262, 264, 286, 291, 305, 308, 309, 323, 335, 343, 345, 358, 372, 373, 387, 391, 392, 400, 412, 414, 415, 425, 428, 431, 433, 434, 435, 436, 447, 451, 453, 464, 466, 482 of SEQ ID NO: 1 or 2, wherein said difference refers to a substitution of the amino acid at that positions by any other amino acid.
 18. The nucleic acid molecule of claim 17, wherein the the amino acid sequence of the encoded mutated PPO polypeptide additionally differs from the wildtype amino acid sequence of a PPO polypeptide at one or more positions corresponding to positions 128, 397, or 420 of SEQ ID NO: 1 or 2, wherein said difference refers to a substitution of the amino acid at that positions by any other amino acid.
 19. The nucleic acid of claim 17, wherein the amino acids at positions corresponding to positions 128, 211, 387, and 420 of SEQ ID NO: 1 or 2 are substituted by any other amino acid.
 20. The nucleic acid of claim 17, wherein the amino acids at positions corresponding to positions 128, 211, and 420 of SEQ ID NO: 1 or 2 are substituted by any other amino acid.
 21. The nucleic acid of claim 17, wherein the amino acids at positions corresponding to positions 128, 210, and 420 of SEQ ID NO: 1 or 2 are substituted by any other amino acid.
 22. The nucleic acid of claim 17, wherein the amino acids at positions corresponding to positions 128, 412, and 420 of SEQ ID NO: 1 or 2 are substituted by any other amino acid.
 23. The nucleic acid of claim 17, wherein the amino acids at positions corresponding to positions 119, 128, and 420 of SEQ ID NO: 1 or 2 are substituted by any other amino acid.
 24. The nucleic acid of claim 17, wherein the amino acids at positions corresponding to positions 211, 397 and 420 of SEQ ID NO: 1 or 2 are substituted by any other amino acid.
 25. The nucleic acid of claim 17, wherein the amino acids at positions corresponding to positions 211, and 420 of SEQ ID NO: 1 or 2 are substituted by any other amino acid
 26. An expression cassette comprising the nucleic acid molecule as defined in claim 17, and a promoter operable in plant cells.
 27. A vector comprising the nucleic acid molecule as defined in claim 17 or the expression cassette.
 28. An isolated, recombinant and/or chemically synthesized mutated PPO polypeptide encoded by the nucleic acid molecule as claimed in claim 17 or a polypeptide having at least 80%, 90%, 95%, 98%, 99% identity to the sequence of SEQ ID NO: 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, 100, 101, 102, 103, 104, 105, 106, 107, 108, 109, 110, 111, 112, 113, 114, 115, 116, 117 or 129, or a variant, paralogue, orthologue or homolog thereof, wherein the amino acid sequence of the mutated PPO polypeptide differs from the wildtype amino acid sequence of a PPO polypeptide at one or more positions corresponding to positions 32, 53, 57, 61, 63, 64, 65, 67, 71, 76, 82, 83, 85, 86, 87, 88, 91, 103, 104, 106, 108, 116, 119, 126, 127, 129, 139, 159, 210, 211, 224, 245, 246, 248, 249, 252, 253, 254, 255, 257, 259, 260, 262, 264, 286, 291, 305, 308, 309, 323, 335, 343, 345, 358, 372, 373, 387, 391, 392, 400, 412, 414, 415, 425, 428, 431, 433, 434, 435, 436, 447, 451, 453, 464, 466, 482 of SEQ ID NO: 1 or 2, wherein said difference refers to a substitution of the amino acid at that positions by any other amino acid.
 29. A method for producing a plant product from the plant of claim 1, the method comprising processing the plant or a plant part thereof to obtain the plant product.
 30. The method of claim 29, wherein the plant product is fodder, seed meal, oil, or seed-treatment-coated seeds.
 31. A plant product obtained from a plant or plant part thereof, wherein the plant or plant part comprises in at least some of its cells a polynucleotide operably linked to a promoter operable in plant cells, the promoter capable of expressing a mutated PPO polypeptide encoded by the polynucleotide, the expression of the mutated PPO polypeptide conferring to the plant tolerance to herbicides, wherein the plant or plant part further exhibits a second or third herbicide-tolerant trait.
 32. The plant product of claim 31, wherein the product is fodder, seed meal, oil, or seed-treatment-coated seed. 